Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

ABSTRACT

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/518,841, which is a divisional of U.S. patent application Ser. No.14/052,176, filed Oct. 11, 2013, now U.S. Pat. No. 8,865,445, which is adivisional of U.S. patent application Ser. No. 12/883,549, filed Sep.16, 2010, now U.S. Pat. No. 8,569,581, which claims the benefit of U.S.Provisional Application No. 61/243,397, filed Sep. 17, 2009, U.S.Provisional Application No. 61/243,531, filed Sep. 18, 2009, U.S.Provisional Application No. 61/243,543, filed Sep. 18, 2009, and U.S.Provisional Application No. 61/243,679, filed Sep. 18, 2009. Thecontents of these applications are fully incorporated herein byreference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under CooperativeAgreement DE-FC36-08G018080 awarded by the Department of Energy. Thegovernment has certain rights in this invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

This application contains a reference to deposits of biologicalmaterial, which deposits are incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to polypeptides having cellulolyticenhancing activity and polynucleotides encoding the polypeptides. Theinvention also relates to nucleic acid constructs, vectors, and hostcells comprising the polynucleotides as well as methods of producing andusing the polypeptides.

Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently linked bybeta-1,4-bonds. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the lignocellulose is converted tofermentable sugars, e.g., glucose, the fermentable sugars are easilyfermented by yeast into ethanol.

WO 2005/074647 discloses polypeptides having cellulolytic enhancingactivity from Thielavia terrestris. WO 2005/074656 disclosespolypeptides having cellulolytic enhancing activity from Thermoascusaurantiacus. WO 2007/089290 discloses polypeptides having cellulolyticenhancing activity from Trichoderma reesei. WO 2009/085935; WO2009/085859; WO 2009/085864; and WO 2009/085868 disclose polypeptideshaving cellulolytic enhancing activity from Myceliophthora thermophila.

There is a need in the art for polypeptides having cellulolyticenhancing activity with improved properties for use in the degradationof cellulosic materials.

The present invention provides polypeptides having cellulolyticenhancing activity and polynucleotides encoding the polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havingcellulolytic enhancing activity selected from the group consisting of:

(a) a polypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 12; at least65% sequence identity to the mature polypeptide of SEQ ID NO: 4; atleast 70% sequence identity to the mature polypeptide SEQ ID NO: 18; atleast 75% sequence identity to the mature polypeptide of SEQ ID NO: 10,SEQ ID NO: 16, or SEQ ID NO: 22; at least 80% sequence identity to themature polypeptide of SEQ ID NO: 8; at least 85% sequence identity tothe mature polypeptide of SEQ ID NO: 14; or at least 90% sequenceidentity to the mature polypeptide of SEQ ID NO: 20;

(b) a polypeptide encoded by a polynucleotide that hybridizes undermedium-high, high, or very high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 11, or SEQ ID NO: 17, (ii) the cDNA sequence containedin the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 11, or SEQ ID NO: 17, or (iii) the full-lengthcomplementary strand of (i) or (ii); high or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 21, (ii)the cDNA sequence contained in the mature polypeptide coding sequence ofSEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO:21, or (iii) the full-length complementary strand of (i) or (ii); orvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 19, (ii) the cDNA sequence contained in themature polypeptide coding sequence of SEQ ID NO: 19, or (iii) thefull-length complementary strand of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide having at least 60%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 5, or SEQ ID NO: 11; at least 65% sequence identity tothe mature polypeptide coding sequence of SEQ ID NO: 3; at least 70%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 17; at least 75% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 9, SEQ ID NO: 15, or SEQ ID NO: 21; at least 80%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 7; at least 85% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 13; or at least 90% sequence identity to themature polypeptide coding sequence of SEQ ID NO: 19; or the cDNAsequences thereof;

(d) a variant comprising a substitution, deletion, and/or insertion ofone or more (several) amino acids of the mature polypeptide of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, orSEQ ID NO: 22; and

(e) a fragment of the polypeptide of (a), (b), (c), or (d) that hascellulolytic enhancing activity.

The present invention also relates to isolated polynucleotides encodingthe polypeptides of the present invention; nucleic acid constructs,recombinant expression vectors, and recombinant host cells comprisingthe polynucleotides; and methods of producing the polypeptides.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity of the present invention.

The present invention also relates to methods of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity of the present invention; (b)fermenting the saccharified cellulosic material with one or more(several) fermenting microorganisms to produce the fermentation product;and (c) recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition in the presence of apolypeptide having cellulolytic enhancing activity of the presentinvention.

The present invention also relates to a polynucleotide encoding a signalpeptide comprising or consisting of amino acids 1 to 17 of SEQ ID NO: 2,amino acids 1 to 19 of SEQ ID NO: 4, amino acids 1 to 17 of SEQ ID NO:6, amino acids 1 to 19 of SEQ ID NO: 8, amino acids 1 to 21 of SEQ IDNO: 10, amino acids 1 to 24 of SEQ ID NO: 12, amino acids 1 to 16 of SEQID NO: 14, amino acids 1 to 18 of SEQ ID NO: 16, amino acids 1 to 22 ofSEQ ID NO: 18, amino acids 1 to 16 of SEQ ID NO: 20, or amino acids 1 to19 of SEQ ID NO: 22, which is operably linked to a gene encoding aprotein; nucleic acid constructs, expression vectors, and recombinanthost cells comprising the polynucleotides; and methods of producing aprotein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the genomic DNA sequence without introns and the deducedamino acid sequence of a gene encoding a Thielavia terrestris NRRL 8126GH61J polypeptide having cellulolytic enhancing activity. Thefull-length genomic DNA sequence with introns is shown in SEQ ID NO: 1and the deduced amino acid sequence is shown in SEQ ID NO: 2.

FIG. 2 shows the genomic DNA sequence without introns and the deducedamino acid sequence of a gene encoding a Thielavia terrestris NRRL 8126GH61K polypeptide having cellulolytic enhancing activity. Thefull-length genomic DNA sequence with introns is shown in SEQ ID NO: 3and the deduced amino acid sequence is shown in SEQ ID NO: 4.

FIG. 3 shows the genomic DNA sequence without introns and the deducedamino acid sequence of a gene encoding a Thielavia terrestris NRRL 8126GH61L polypeptide having cellulolytic enhancing activity. Thefull-length genomic DNA sequence with introns is shown in SEQ ID NO: 5and the deduced amino acid sequence is shown in SEQ ID NO: 6.

FIG. 4 shows hydrolysis of pretreated corn stover (PCS) with aTrichoderma reesei cellulase mixture in the presence of varyingconcentrations of Thielavia terrestris NRRL 8126 GH61J, GH61K, and GH61Lpolypeptides having cellulolytic enhancing activity.

FIG. 5 shows a restriction map of pSMai197.

FIG. 6 shows the genomic DNA sequence and the deduced amino acidsequence of a gene encoding a Thielavia terrestris NRRL 8126 GH61Mpolypeptide having cellulolytic enhancing activity (SEQ ID NOs: 7 and 8,respectively).

FIG. 7 shows hydrolysis of pretreated corn stover (PCS) with aTrichoderma reesei cellulase mixture in the presence of varyingconcentrations of Thielavia terrestris NRRL 8126 GH61M polypeptidehaving cellulolytic enhancing activitty.

FIG. 8 shows the genomic DNA sequence and the deduced amino acidsequence of a gene encoding a Thielavia terrestris NRRL 8126 GH61Npolypeptide having cellulolytic enhancing activity (SEQ ID NOs: 9 and10, respectively).

FIG. 9 shows hydrolysis of pretreated corn stover (PCS) with aTrichoderma reesei cellulase mixture in the presence of varyingconcentrations of Thielavia terrestris NRRL 8126 GH61N polypeptidehaving cellulolytic enhancing activity.

FIG. 10 shows the genomic DNA sequence and the deduced amino acidsequence of a gene encoding a Thielavia terrestris NRRL 8126 GH61Opolypeptide having cellulolytic enhancing activity (SEQ ID NOs: 11 and12, respectively).

FIG. 11 shows the genomic DNA sequence and the deduced amino acidsequence of a gene encoding a Thielavia terrestris NRRL 8126 GH61Ppolypeptide having cellulolytic enhancing activity (SEQ ID NOs: 13 and14, respectively).

FIG. 12 shows the genomic DNA sequence and the deduced amino acidsequence of a gene encoding a Thielavia terrestris NRRL 8126 GH61Rpolypeptide having cellulolytic enhancing activity (SEQ ID NOs: 15 and16, respectively).

FIG. 13 shows the genomic DNA sequence and the deduced amino acidsequence of a gene encoding a Thielavia terrestris NRRL 8126 GH61Spolypeptide having cellulolytic enhancing activity (SEQ ID NOs: 17 and18, respectively).

FIG. 14 shows the genomic DNA sequence and the deduced amino acidsequence of a gene encoding a Thielavia terrestris NRRL 8126 GH61Tpolypeptide having cellulolytic enhancing activity (SEQ ID NOs: 19 and20, respectively).

FIG. 15 shows the genomic DNA sequence and the deduced amino acidsequence of a gene encoding a Thielavia terrestris NRRL 8126 GH61Upolypeptide having cellulolytic enhancing activity (SEQ ID NOs: 21 and22, respectively).

Definitions

Polypeptide having cellulolytic enhancing activity: The term“polypeptide having cellulolytic enhancing activity” means a GH61polypeptide that enhances the hydrolysis of a cellulosic material byenzyme having cellulolytic activity. For purposes of the presentinvention, cellulolytic enhancing activity is determined by measuringthe increase in reducing sugars or the increase of the total ofcellobiose and glucose from the hydrolysis of a cellulosic material bycellulolytic enzyme under the following conditions: 1-50 mg of totalprotein/g of cellulose in PCS, wherein total protein is comprised of50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/w protein of aGH61 polypeptide having cellulolytic enhancing activity for 1-7 days at50° C. compared to a control hydrolysis with equal total protein loadingwithout cellulolytic enhancing activity (1-50 mg of cellulolyticprotein/g of cellulose in PCS). In a preferred aspect, a mixture ofCELLUCLAST® 1.5 L (Novozymes A/S, Bagsvrd, Denmark) in the presence of2-3% of total protein weight Aspergillus oryzae beta-glucosidase(recombinantly produced in Aspergillus oryzae according to WO 02/095014)or 2-3% of total protein weight Aspergillus fumigatus beta-glucosidase(recombinantly produced in Aspergillus oryzae as described in WO2002/095014) of cellulase protein loading is used as the source of thecellulolytic activity.

The GH61 polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, more preferably at least 1.05-fold, more preferably at least1.10-fold, more preferably at least 1.25-fold, more preferably at least1.5-fold, more preferably at least 2-fold, more preferably at least3-fold, more preferably at least 4-fold, more preferably at least5-fold, even more preferably at least 10-fold, and most preferably atleast 20-fold.

The polypeptides of the present invention have at least 20%, e.g., atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, and at least 100% of the cellulolytic enhancingactivity of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ IDNO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic activity include: (1)measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman N21filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No 1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-20mg of cellulolytic enzyme protein/g of cellulose in PCS for 3-7 days at50° C. compared to a control hydrolysis without addition of cellulolyticenzyme protein. Typical conditions are 1 ml reactions, washed orunwashed PCS, 5% insoluble solids, 50 mM sodium acetate pH 5, 1 mMMnSO₄, 50° C., 72 hours, sugar analysis by AMINEX® HPX-87H column(Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Endoglucanase: The term “endoglucanase” means anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4),which catalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages incellulose, cellulose derivatives (such as carboxymethyl cellulose andhydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3glucans such as cereal beta-D-glucans or xyloglucans, and other plantmaterial containing cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91), which catalyzes thehydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellooligosaccharides, or any beta-1,4-linked glucose containingpolymer, releasing cellobiose from the reducing or non-reducing ends ofthe chain (Teeri, 1997, Crystalline cellulose degradation: New insightinto the function of cellobiohydrolases, Trends in Biotechnology 15:160-167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: whyso efficient on crystalline cellulose?, Biochem. Soc. Trans. 26:173-178). For purposes of the present invention, cellobiohydrolaseactivity is determined according to the procedures described by Lever etal., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBSLetters, 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters,187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. Inthe present invention, the Lever et al. method can be employed to assesshydrolysis of cellulose in corn stover, while the methods of vanTilbeurgh et al. and Tomme et al. can be used to determine thecellobiohydrolase activity on a fluorescent disaccharide derivative,4-methylumbelliferyl-p-D-lactoside.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21), which catalyzes the hydrolysis ofterminal non-reducing beta-D-glucose residues with the release ofbeta-D-glucose. For purposes of the present invention, beta-glucosidaseactivity is determined according to the basic procedure described byVenturi et al., 2002, Extracellular beta-D-glucosidase from Chaetomiumthermophilum var. coprophilum: production, purification and somebiochemical properties, J. Basic Microbiol. 42: 55-66. One unit ofbeta-glucosidase is defined as 1.0 μmole of p-nitrophenolate anionproduced per minute at 25° C., pH 4.8 from 1 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodiumcitrate containing 0.01% TWEEN® 20.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (several) enzymes thathydrolyze a hemicellulosic material. See, for example, Shallom, D. andShoham, Y. Microbial hemicellulases. Current Opinion In Microbiology,2003, 6(3): 219-228). Hemicellulases are key components in thedegradation of plant biomass. Examples of hemicellulases include, butare not limited to, an acetylmannan esterase, an acetyxylan esterase, anarabinanase, an arabinofuranosidase, a coumaric acid esterase, aferuloyl esterase, a galactosidase, a glucuronidase, a glucuronoylesterase, a mannanase, a mannosidase, a xylanase, and a xylosidase. Thesubstrates of these enzymes, the hemicelluloses, are a heterogeneousgroup of branched and linear polysaccharides that are bound via hydrogenbonds to the cellulose microfibrils in the plant cell wall, crosslinkingthem into a robust network. Hemicelluloses are also covalently attachedto lignin, forming together with cellulose a highly complex structure.The variable structure and organization of hemicelluloses require theconcerted action of many enzymes for its complete degradation. Thecatalytic modules of hemicellulases are either glycoside hydrolases(GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases (CEs),which hydrolyze ester linkages of acetate or ferulic acid side groups.These catalytic modules, based on homology of their primary sequence,can be assigned into GH and CE families marked by numbers. Somefamilies, with overall similar fold, can be further grouped into clans,marked alphabetically (e.g., GH-A). A most informative and updatedclassification of these and other carbohydrate active enzymes isavailable on the Carbohydrate-Active Enzymes (CAZy) database.Hemicellulolytic enzyme activities can be measured according to Ghoseand Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, Recent progress in the assays of xylanolytic enzymes, 2006,Journal of the Science of Food and Agriculture 86(11): 1636-1647;Spanikova and Biely, 2006, Glucuronoyl esterase—Novel carbohydrateesterase produced by Schizophyllum commune, FEBS Letters 580(19):4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek,1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctionalbeta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. The most common total xylanolytic activity assay is basedon production of reducing sugars from polymeric 4-O-methylglucuronoxylan as described in Bailey, Biely, Poutanen, 1992,Interlaboratory testing of methods for assay of xylanase activity,Journal of Biotechnology 23(3): 257-270. Xylanase activity can also bedetermined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON®X-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unit ofxylanase activity is defined as 1.0 μmole of azurine produced per minuteat 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mMsodium phosphate pH 6 buffer.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, A new reaction for colorimetric determination ofcarbohydrates, Anal. Biochem 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. For purposes of the present invention, xylanaseactivity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01%TRITON® X-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unitof xylanase activity is defined as 1.0 μmole of azurine produced perminute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate pH 6 buffer.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides, to remove successive D-xylose residuesfrom the non-reducing termini. For purposes of the present invention,one unit of beta-xylosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20.

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of thepresent invention, acetylxylan esterase activity is determined using 0.5mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20. One unit of acetylxylan esterase is definedas the amount of enzyme capable of releasing 1 μmole of p-nitrophenolateanion per minute at pH 5, 25° C.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl)group from an esterified sugar, which is usually arabinose in “natural”substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloylesterase is also known as ferulic acid esterase, hydroxycinnamoylesterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, orFAE-II. For purposes of the present invention, feruloyl esteraseactivity is determined using 0.5 mM p-nitrophenylferulate as substratein 50 mM sodium acetate pH 5.0. One unit of feruloyl esterase equals theamount of enzyme capable of releasing 1 μmole of p-nitrophenolate anionper minute at pH 5, 25° C.

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. For purposes of the present invention, alpha-glucuronidaseactivity is determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of alpha-glucuronidase equals the amount of enzymecapable of releasing 1 μmole of glucuronic or 4-O-methylglucuronic acidper minute at pH 5, 40° C.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of thepresent invention, alpha-L-arabinofuranosidase activity is determinedusing 5 mg of medium viscosity wheat arabinoxylan (MegazymeInternational Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40°C. followed by arabinose analysis by AMINEX® HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, herbaceous material,agricultural residue, forestry residue, municipal solid waste, wastepaper, and pulp and paper mill residue (see, for example, Wiselogel etal., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor),pp.105-118, Taylor & Francis, Washington D.C.; Wyman, 1994, BioresourceTechnology 50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology24/25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion ofLignocellulosics, in Advances in Biochemical Engineering/Biotechnology,T. Scheper, managing editor, Volume 65, pp.23-40, Springer-Verlag, NewYork). It is understood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is lignocellulose, which comprises cellulose,hemicellulose, and lignin.

In one aspect, the cellulosic material is herbaceous material. Inanother aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is forestry residue. In anotheraspect, the cellulosic material is municipal solid waste. In anotheraspect, the cellulosic material is waste paper. In another aspect, thecellulosic material is pulp and paper mill residue.

In another aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is corn fiber. In another aspect, thecellulosic material is corn cob. In another aspect, the cellulosicmaterial is orange peel. In another aspect, the cellulosic material isrice straw. In another aspect, the cellulosic material is wheat straw.In another aspect, the cellulosic material is switch grass. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is bagasse.

In another aspect, the cellulosic material is microcrystallinecellulose. In another aspect, the cellulosic material is bacterialcellulose. In another aspect, the cellulosic material is algalcellulose. In another aspect, the cellulosic material is cotton linter.In another aspect, the cellulosic material is amorphous phosphoric-acidtreated cellulose. In another aspect, the cellulosic material is filterpaper.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Pretreated corn stover: The term “PCS” or “Pretreated Corn Stover” meansa cellulosic material derived from corn stover by treatment with heatand dilute sulfuric acid.

Xylan-containing material: The term “xylan-containing material” meansany material comprising a plant cell wall polysaccharide containing abackbone of beta-(1-4)-linked xylose residues. Xylans of terrestrialplants are heteropolymers possessing a beta-(1-4)-D-xylopyranosebackbone, which is branched by short carbohydrate chains. They compriseD-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or variousoligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose,and D-glucose. Xylan-type polysaccharides can be divided into homoxylansand heteroxylans, which include glucuronoxylans,(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, andcomplex heteroxylans. See, for example, Ebringerova et al., 2005, Adv.Polym. Sci. 186: 1-67.

In the methods of the present invention, any material containing xylanmay be used. In a preferred aspect, the xylan-containing material islignocellulose.

Isolated or Purified: The term “isolated” or “purified” means apolypeptide or polynucleotide that is removed from at least onecomponent with which it is naturally associated. For example, apolypeptide may be at least 1% pure, e.g., at least 5% pure, at least10% pure, at least 20% pure, at least 40% pure, at least 60% pure, atleast 80% pure, at least 90% pure, or at least 95% pure, as determinedby SDS-PAGE, and a polynucleotide may be at least 1% pure, e.g., atleast 5% pure, at least 10% pure, at least 20% pure, at least 40% pure,at least 60% pure, at least 80% pure, at least 90% pure, or at least 95%pure, as determined by agarose electrophoresis.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 18 to 246 of SEQ ID NO: 2 based on theSignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6) thatpredicts amino acids 1 to 17 of SEQ ID NO: 2 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 20 to 334 of SEQID NO: 4 based on the SignalP program that predicts amino acids 1 to 19of SEQ ID NO: 4 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 18 to 227 of SEQ ID NO: 6 based on theSignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 6 are asignal peptide. In another aspect, the mature polypeptide is amino acids20 to 223 of SEQ ID NO: 8 based on the SignalP program that predictsamino acids 1 to 19 of SEQ ID NO: 8 are a signal peptide. In anotheraspect, the mature polypeptide is amino acids 22 to 368 of SEQ ID NO: 10based on the SignalP program that predicts amino acids 1 to 21 of SEQ IDNO: 10 are a signal peptide. In another aspect, the mature polypeptideis amino acids 25 to 330 of SEQ ID NO: 12 based on the SignalP programthat predicts amino acids 1 to 24 of SEQ ID NO: 12 are a signal peptide.In another aspect, the mature polypeptide is amino acids 17 to 236 ofSEQ ID NO: 14 based on the SignalP program that predicts amino acids 1to 16 of SEQ ID NO: 14 are a signal peptide. In another aspect, themature polypeptide is amino acids 19 to 250 of SEQ ID NO: 16 based onthe SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 16are a signal peptide. In another aspect, the mature polypeptide is aminoacids 23 to 478 of SEQ ID NO: 18 based on the SignalP program thatpredicts amino acids 1 to 22 of SEQ ID NO: 18 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 17 to 230 of SEQID NO: 20 based on the SignalP program that predicts amino acids 1 to 16of SEQ ID NO: 20 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 20 to 257 of SEQ ID NO: 22 based on theSignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 22 are asignal peptide. It is known in the art that a host cell may produce amixture of two of more different mature polypeptides (i.e., with adifferent C-terminal and/or N-terminal amino acid) expressed by the samepolynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving cellulolytic enhancing activity. In one aspect, the maturepolypeptide coding sequence is nucleotides 52 to 875 of SEQ ID NO: 1based on the SignalP program (Nielsen et al., 1997, supra) that predictsnucleotides 1 to 51 of SEQ ID NO: 1 encode a signal peptide. In anotheraspect, the mature polypeptide coding sequence is the cDNA sequencecontained in nucleotides 52 to 875 of SEQ ID NO: 1. In another aspect,the mature polypeptide coding sequence is nucleotides 58 to 1250 of SEQID NO: 3 based on the SignalP program that predicts nucleotides 1 to 57of SEQ ID NO: 3 encode a signal peptide. In another aspect, the maturepolypeptide coding sequence is the cDNA sequence contained innucleotides 58 to 1250 of SEQ ID NO: 3. In another aspect, the maturepolypeptide coding sequence is nucleotides 52 to 795 of SEQ ID NO: 5based on the SignalP program that predicts nucleotides 1 to 51 of SEQ IDNO: 5 encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is the cDNA sequence contained in nucleotides 52 to 795of SEQ ID NO: 5. In another aspect, the mature polypeptide codingsequence is nucleotides 58 to 974 of SEQ ID NO: 7 based on the SignalPprogram that predicts nucleotides 1 to 57 of SEQ ID NO: 7 encode asignal peptide. In another aspect, the mature polypeptide codingsequence is the cDNA sequence contained in nucleotides 58 to 974 of SEQID NO: 7. In another aspect, the mature polypeptide coding sequence isnucleotides 64 to 1104 of SEQ ID NO: 9 based on the SignalP program thatpredicts nucleotides 1 to 63 of SEQ ID NO: 9 encode a signal peptide. Inanother aspect, the mature polypeptide coding sequence is the cDNAsequence contained in nucleotides 64 to 1104 of SEQ ID NO: 9. In anotheraspect, the mature polypeptide coding sequence is nucleotides 73 to 990of SEQ ID NO: 11 based on the SignalP program that predicts nucleotides1 to 72 of SEQ ID NO: 11 encode a signal peptide. In another aspect, themature polypeptide coding sequence is the cDNA sequence contained innucleotides 73 to 990 of SEQ ID NO: 11. In another aspect, the maturepolypeptide coding sequence is nucleotides 49 to 1218 of SEQ ID NO: 13based on the SignalP program that predicts nucleotides 1 to 48 of SEQ IDNO: 13 encode a signal peptide. In another aspect, the maturepolypeptide coding sequence is the cDNA sequence contained innucleotides 49 to 1218 of SEQ ID NO: 13. In another aspect, the maturepolypeptide coding sequence is nucleotides 55 to 930 of SEQ ID NO: 15based on the SignalP program that predicts nucleotides 1 to 54 of SEQ IDNO: 15 encode a signal peptide. In another aspect, the maturepolypeptide coding sequence is the cDNA sequence contained innucleotides 55 to 930 of SEQ ID NO: 15. In another aspect, the maturepolypeptide coding sequence is nucleotides 67 to 1581 of SEQ ID NO: 17based on the SignalP program that predicts nucleotides 1 to 66 of SEQ IDNO: 17 encode a signal peptide. In another aspect, the maturepolypeptide coding sequence is the cDNA sequence contained innucleotides 67 to 1581 of SEQ ID NO: 17. In another aspect, the maturepolypeptide coding sequence is nucleotides 49 to 865 of SEQ ID NO: 19based on the SignalP program that predicts nucleotides 1 to 48 of SEQ IDNO: 19 encode a signal peptide. In another aspect, the maturepolypeptide coding sequence is the cDNA sequence contained innucleotides 49 to 865 of SEQ ID NO: 19. In another aspect, the maturepolypeptide coding sequence is nucleotides 58 to 1065 of SEQ ID NO: 21based on the SignalP program that predicts nucleotides 1 to 57 of SEQ IDNO: 21 encode a signal peptide. In another aspect, the maturepolypeptide coding sequence is the cDNA sequence contained innucleotides 58 to 1065 of SEQ ID NO: 21.

Sequence Identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the—nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the—nobrief option)is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Fragment: The term “fragment” means a polypeptide having one or more(several) amino acids deleted from the amino and/or carboxyl terminus ofa mature polypeptide; wherein the fragment has cellulolytic enhancingactivity. In a one aspect, a fragment contains at least 190 amino acidresidues, e.g., at least 200 amino acid residues or at least 210 aminoacid residues of the mature polypeptide of SEQ ID NO: 2. In anotheraspect, a fragment contains at least 265 amino acid residues, e.g., atleast 280 amino acid residues or at least 295 amino acid residues of themature polypeptide of SEQ ID NO: 4. In another aspect, a fragmentcontains at least 180 amino acid residues, e.g., at least 190 amino acidresidues or at least 200 amino acid residues of the mature polypeptideof SEQ ID NO: 6. In another aspect, a fragment contains at least 170amino acid residues, e.g., at least 180 amino acid residues or at least190 amino acid residues, of the mature polypeptide of SEQ ID NO: 8. Inanother aspect, a fragment contains at least 305 amino acid residues,e.g., at least 330 amino acid residues or at least 335 amino acidresidues of the mature polypeptide of SEQ ID NO: 10. In another aspect,a fragment contains at least 255 amino acid residues, e.g., at least 270amino acid residues or at least 285 amino acid residues of the maturepolypeptide of SEQ ID NO: 12. In another aspect, a fragment contains atleast 190 amino acid residues, e.g., at least 200 amino acid residues orat least 210 amino acid residues of the mature polypeptide of SEQ ID NO:14. In another aspect, a fragment contains at least 200 amino acidresidues, e.g., at least 210 amino acid residues or at least 220 aminoacid residues of the mature polypeptide of SEQ ID NO: 16. In anotheraspect, a fragment contains at least 390 amino acid residues, e.g., atleast 410 amino acid residues or at least 430 amino acid residues of themature polypeptide of SEQ ID NO: 18. In another aspect, a fragmentcontains at least 180 amino acid residues, e.g., at least 190 amino acidresidues or at least 200 amino acid residues of the mature polypeptideof SEQ ID NO: 20. In another aspect, a fragment contains at least 210amino acid residues, e.g., at least 220 amino acid residues or at least230 amino acid residues of the mature polypeptide of SEQ ID NO: 22.

Subsequence: The term “subsequence” means a polynucleotide having one ormore (several) nucleotides deleted from the 5′ and/or 3′ end of a maturepolypeptide coding sequence; wherein the subsequence encodes a fragmenthaving cellulolytic enhancing activity. In one aspect, a subsequencecontains at least 570 nucleotides, e.g., at least 600 nucleotides or atleast 630 nucleotides of the mature polypeptide coding sequence of SEQID NO: 1. In another preferred aspect, a subsequence contains at least795 nucleotides, e.g., at least 840 nucleotides or at least 885nucleotides of the mature polypeptide coding sequence of SEQ ID NO: 3.In another preferred aspect, a subsequence contains at least 540nucleotides, e.g., at least 570 nucleotides or at least 600 nucleotidesof the mature polypeptide coding sequence of SEQ ID NO: 5. In apreferred aspect, a subsequence contains at least 510 nucleotides, e.g.,at least 540 nucleotides or at least 570 nucleotides of the maturepolypeptide coding sequence of SEQ ID NO: 7. In another aspect, asubsequence contains at least 915 nucleotides, e.g., at least 960nucleotides or at least 1005 nucleotides of the mature polypeptidecoding sequence of SEQ ID NO: 9. In another preferred aspect, asubsequence contains at least 765 nucleotides, e.g., at least 810nucleotides or at least 855 nucleotides of the mature polypeptide codingsequence of SEQ ID NO: 11. In another preferred aspect, a subsequencecontains at least 570 nucleotides, e.g., at least 600 nucleotides or atleast 630 nucleotides of the mature polypeptide coding sequence of SEQID NO: 13. In another preferred aspect, a subsequence contains at least600 nucleotides, e.g., at least 630 nucleotides or at least 660nucleotides of the mature polypeptide coding sequence of SEQ ID NO: 15.In another preferred aspect, a subsequence contains at least 1170nucleotides, e.g., at least 1230 nucleotides or at least 1290nucleotides of the mature polypeptide coding sequence of SEQ ID NO: 17.In another preferred aspect, a subsequence contains at least 540nucleotides, e.g., at least 570 nucleotides or at least 600 nucleotidesof the mature polypeptide coding sequence of SEQ ID NO: 19. In anotherpreferred aspect, a subsequence contains at least 630 nucleotides, e.g.,at least 660 nucleotides or at least 690 nucleotides of the maturepolypeptide coding sequence of SEQ ID NO: 21.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which usually begins with the ATG start codon oralternative start codons such as GTG and TTG and ends with a stop codonsuch as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA,synthetic, or recombinant polynucleotide.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic cell. cDNA lacks intron sequences that may be presentin the corresponding genomic DNA. The initial, primary RNA transcript isa precursor to mRNA that is processed through a series of steps,including splicing, before appearing as mature spliced mRNA.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic. The term nucleic acid construct issynonymous with the term “expression cassette” when the nucleic acidconstruct contains the control sequences required for expression of acoding sequence of the present invention.

Control sequences: The term “control sequences” means all componentsnecessary for the expression of a polynucleotide encoding a polypeptideof the present invention. Each control sequence may be native or foreignto the polynucleotide encoding the polypeptide or native or foreign toeach other. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs the expression of the coding sequence.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to additional nucleotides thatprovide for its expression.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, and the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Variant: The term “variant” means a polypeptide having cellulolyticenhancing activity comprising an alteration, i.e., a substitution,insertion, and/or deletion of one or more (several) amino acid residuesat one or more (several) positions. A substitution means a replacementof an amino acid occupying a position with a different amino acid; adeletion means removal of an amino acid occupying a position; and aninsertion means adding one or more (several) amino acids, e.g., 1-5amino acids, adjacent to an amino acid occupying a position.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides Having CellulolyticEnhancing Activity

The present invention relates to isolated polypeptides havingcellulolytic enhancing activity selected from the group consisting of:

(a) a polypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 12; at least65% sequence identity to the mature polypeptide of SEQ ID NO: 4; atleast 70% sequence identity to the mature polypeptide of SEQ ID NO: 18;at least 75% sequence identity to the mature polypeptide of SEQ ID NO:10, SEQ ID NO: 16, or SEQ ID NO: 22; at least 80% sequence identity tothe mature polypeptide of SEQ ID NO: 8; at least 85% sequence identityto the mature polypeptide of SEQ ID NO: 14; or at least 90% sequenceidentity to the mature polypeptide of SEQ ID NO: 20;

(b) a polypeptide encoded by a polynucleotide that hybridizes undermedium-high, high, or very high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 11, or SEQ ID NO: 17, (ii) the cDNA sequence containedin the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 11, or SEQ ID NO: 17, or (iii) the full-lengthcomplementary strand of (i) or (ii); high or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 21, (ii)the cDNA sequence contained in the mature polypeptide coding sequence ofSEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO:21, or (iii) the full-length complementary strand of (i) or (ii); orvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 19, (ii) the cDNA sequence contained in themature polypeptide coding sequence of SEQ ID NO: 19, or (iii) thefull-length complementary strand of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide having at least 60%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 5, or SEQ ID NO: 11; at least 65% sequence identity tothe mature polypeptide coding sequence of SEQ ID NO: 3; at least 70%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 17; at least 75% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 9, SEQ ID NO: 15, or SEQ ID NO: 21; at least 80%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 7; at least 85% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 13; or at least 90% sequence identity to themature polypeptide coding sequence of SEQ ID NO: 19; or the cDNAsequences thereof;

(d) a variant comprising a substitution, deletion, and/or insertion ofone or more (several) amino acids of the mature polypeptide of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, orSEQ ID NO: 22; and

(e) a fragment of a polypeptide of (a), (b), (c), or (d) that hascellulolytic enhancing activity.

The present invention relates to isolated polypeptides having a sequenceidentity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 6, or SEQID NO: 12 of at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%; the mature polypeptide of SEQ ID NO: 4 of at least 65%,e.g., at least 70%, at least 75%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100%; the mature polypeptide of SEQID NO: 18 of at least 70%, e.g., at least 75%, at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100%; the maturepolypeptide of SEQ ID NO: 10, SEQ ID NO: 16, or SEQ ID NO: 22 of atleast 75%, e.g., at least 80%, at least 81%, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%; the mature polypeptide of SEQ ID NO: 8 of at least80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%; the mature polypeptide of SEQ ID NO: 14 of at least 85%, e.g., atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%; or themature polypeptide of SEQ ID NO: 20 at least 90%, e.g., at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%, which have cellulolyticenhancing activity.

In one aspect, the polypeptides differ by no more than ten amino acids,e.g., by five amino acids, by four amino acids, by three amino acids, bytwo amino acids, and by one amino acid from the mature polypeptide ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, or SEQ ID NO: 22.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22 or an allelic variantthereof; or is a fragment thereof having cellulolytic enhancingactivity. In another aspect, the polypeptide comprises or consists ofthe mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16,SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22. In another preferredaspect, the polypeptide comprises or consists of amino acids 18 to 246of SEQ ID NO: 2. In another preferred aspect, the polypeptide comprisesor consists of amino acids 20 to 334 of SEQ ID NO: 4. In anotherpreferred aspect, the polypeptide comprises or consists of amino acids18 to 227 of SEQ ID NO: 6. In another preferred aspect, the polypeptidecomprises or consists of amino acids 20 to 223 of SEQ ID NO: 8. Inanother preferred aspect, the polypeptide comprises or consists of aminoacids 22 to 368 of SEQ ID NO: 10. In another preferred aspect, thepolypeptide comprises or consists of amino acids 25 to 330 of SEQ ID NO:12. In another preferred aspect, the polypeptide comprises or consistsof amino acids 17 to 236 of SEQ ID NO: 14. In another preferred aspect,the polypeptide comprises or consists of amino acids 19 to 250 of SEQ IDNO: 16. In another preferred aspect, the polypeptide comprises orconsists of amino acids 23 to 478 of SEQ ID NO: 18. In another preferredaspect, the polypeptide comprises or consists of amino acids 17 to 230of SEQ ID NO: 20. In another preferred aspect, the polypeptide comprisesor consists of amino acids 20 to 257 of SEQ ID NO: 22.

The present invention also relates to isolated polypeptides havingcellulolytic enhancing activity that are encoded by polynucleotides thathybridize under medium-high, high, or very high stringency conditionswith (i) the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5, SEQ ID NO: 11, or SEQ ID NO: 17, (ii) the cDNAsequence contained in the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 11, or SEQ ID NO: 17, or(iii) the full-length complementary strand of (i) or (ii); high or veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, orSEQ ID NO: 21, (ii) the cDNA sequence contained in the maturepolypeptide coding sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:13, SEQ ID NO: 15, or SEQ ID NO: 21, or (iii) the full-lengthcomplementary strand of (i) or (ii); or very high stringency conditionswith (i) the mature polypeptide coding sequence of SEQ ID NO: 19, (ii)the cDNA sequence contained in the mature polypeptide coding sequence ofSEQ ID NO: 19, or (iii) the full-length complementary strand of (i) or(ii) (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, MolecularCloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, or SEQ ID NO: 21, or a subsequence thereof, aswell as the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ IDNO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22, or a fragmentthereof, may be used to design nucleic acid probes to identify and cloneDNA encoding polypeptides having cellulolytic enhancing activity fromstrains of different genera or species according to methods well knownin the art. In particular, such probes can be used for hybridizationwith the genomic DNA or cDNA of the genus or species of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least14, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having cellulolytic enhancing activity. Genomic orother DNA from such other strains may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that is homologous with SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, or SEQ IDNO: 21, ora subsequence thereof, the carrier material is preferably usedin a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, or SEQ ID NO: 21; the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, orSEQ ID NO: 21; the cDNA sequence contained in the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, or SEQ ID NO: 21; the full-length complementarystrands thereof; or a subsequence thereof; under very low to very highstringency conditions. Molecules to which the nucleic acid probehybridizes under these conditions can be detected using, for example,X-ray film.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,SEQ ID NO: 19, or SEQ ID NO: 21, or the cDNA sequences thereof. Inanother aspect, the nucleic acid probe is nucleotides 52 to 875 of SEQID NO: 1, nucleotides 58 to 1250 of SEQ ID NO: 3, nucleotides 52 to 795of SEQ ID NO: 5, nucleotides 58 to 974 of SEQ ID NO: 7, nucleotides 64to 1104 of SEQ ID NO: 9, nucleotides 73 to 990 of SEQ ID NO: 11,nucleotides 49 to 1218 of SEQ ID NO: 13, nucleotides 55 to 930 of SEQ IDNO: 15, nucleotides 67 to 1581 of SEQ ID NO: 17, nucleotides 49 to 865of SEQ ID NO: 19, or nucleotides 58 to 1065 of SEQ ID NO: 21. In anotheraspect, the nucleic acid probe is a polynucleotide that encodes thepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 20, or SEQ ID NO: 22, or the mature polypeptides thereof;or fragments thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ IDNO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQID NO: 19, or SEQ ID NO: 21, or the cDNA sequences thereof. In anotheraspect, the nucleic acid probe is the polynucleotide sequence containedin plasmid pSMai216 which is contained in E. coli NRRL B-50301, whereinthe polynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another aspect, the nucleic acidprobe is the mature polypeptide coding region contained in plasmidpSMai21 which is contained in E. coli NRRL B-50301. In another aspect,the nucleic acid probe is the polynucleotide sequence contained inplasmid pSMAi217 which is contained in E. coli NRRL B-50302, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another aspect, the nucleic acidprobe is the mature polypeptide coding region contained in plasmidpSMai217 which is contained in E. coli NRRL B-50302. In another aspect,the nucleic acid probe is the polynucleotide sequence contained inplasmid pSMai218 which is contained in E. coli NRRL B-50303, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another aspect, the nucleic acidprobe is the mature polypeptide coding region contained in plasmidpSMai218 which is contained in E. coli NRRL B-50303. In another aspect,the nucleic acid probe is the polynucleotide sequence contained inplasmid pSMai213 which is contained in E. coli NRRL B-50300, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another aspect, the nucleic acidprobe is the mature polypeptide coding region contained in plasmidpSMai213 which is contained in E. coli NRRL B-50300. In another aspect,the nucleic acid probe is the polynucleotide sequence contained inplasmid pAG68 which is contained in E. coli NRRL B-50320, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another aspect, the nucleic acidprobe is the mature polypeptide coding region contained in plasmid pAG68which is contained in E. coli NRRL B-50320. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in plasmidpAG69 which is contained in E. coli NRRL B-50321, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another aspect, the nucleic acidprobe is the mature polypeptide coding region contained in plasmid pAG69which is contained in E. coli NRRL B-50321. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in plasmidpAG75 which is contained in E. coli NRRL B-50322, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another aspect, the nucleic acidprobe is the mature polypeptide coding region contained in plasmid pAG75which is contained in E. coli NRRL B-50322. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in plasmidpAG76 which is contained in E. coli NRRL B-50323, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another aspect, the nucleic acidprobe is the mature polypeptide coding region contained in plasmid pAG76which is contained in E. coli NRRL B-50323. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in plasmidpAG77 which is contained in E. coli NRRL B-50324, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another aspect, the nucleic acidprobe is the mature polypeptide coding region contained in plasmid pAG77which is contained in E. coli NRRL B-50324. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in plasmidpAG78 which is contained in E. coli NRRL B-50325, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another aspect, the nucleic acidprobe is the mature polypeptide coding region contained in plasmid pAG78which is contained in E. coli NRRL B-50325. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in plasmidpAG79 which is contained in E. coli NRRL B-50326, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another aspect, the nucleic acidprobe is the mature polypeptide coding region contained in plasmid pAG79which is contained in E. coli NRRL B-50326.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5× SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and either 25% formamide for very lowand low stringencies, 35% formamide for medium and medium-highstringencies, or 50% formamide for high and very high stringencies,following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed three times each for15 minutes using 2× SSC, 0.2% SDS at 45° C. (very low stringency), at50° C. (low stringency), at 55° C. (medium stringency), at 60° C.(medium-high stringency), at 65° C. (high stringency), and at 70° C.(very high stringency).

For short probes of about 15 nucleotides to about 70 nucleotides inlength, stringency conditions are defined as prehybridization andhybridization at about 5° C. to about 10° C. below the calculated T_(m)using the calculation according to Bolton and McCarthy (1962, Proc.Natl. Acad. Sci. USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6mM EDTA, 0.5% NP-40, 1× Denhardt's solution, 1 mM sodium pyrophosphate,1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA perml following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed once in 6× SCC plus0.1% SDS for 15 minutes and twice each for 15 minutes using 6× SSC at 5°C. to 10° C. below the calculated T_(m).

The present invention also relates to isolated polypeptides havingcellulolytic enhancing activity encoded by polynucleotides having asequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 5, or SEQ ID NO: 11 of at least 60%,at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100%; the maturepolypeptide of SEQ ID NO: 3 of at least 65%, e.g., at least 70%, atleast 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%; the mature polypeptide of SEQ ID NO: 17 of at least70%, e.g., at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%; the mature polypeptide of SEQ ID NO:9, SEQ ID NO: 15, or SEQ ID NO: 21 of at least 75%, e.g., at least 80%,at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%; the maturepolypeptide of SEQ ID NO: 7 of at least 80%, e.g., at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%; the mature polypeptideof SEQ ID NO: 13 of at least 85%, e.g., at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%; or the mature polypeptide of SEQ IDNO: 19 at least 90%, e.g., at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%.

The present invention also relates to variants comprising asubstitution, deletion, and/or insertion of one or more (or several)amino acids of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22, or homologoussequences thereof. Preferably, amino acid changes are of a minor nature,that is conservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for cellulolytic enhancingactivity to identify amino acid residues that are critical to theactivity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem.271: 4699-4708. The active site of the enzyme or other biologicalinteraction can also be determined by physical analysis of structure, asdetermined by such techniques as nuclear magnetic resonance,crystallography, electron diffraction, or photoaffinity labeling, inconjunction with mutation of putative contact site amino acids. See, forexample, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992,J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred fromanalysis of identities with polypeptides that are related to the parentpolypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22 is not morethan 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9.

The polypeptide may be hybrid polypeptide in which a portion of onepolypeptide is fused at the N-terminus or the C-terminus of a portion ofanother polypeptide.

The polypeptide may be a fused polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusedpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator. Fusion proteins may also be constructedusing intein technology in which fusions are createdpost-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawsonet al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Sources of Polypeptides Having Cellulolytic Enhancing Activity

A polypeptide having cellulolytic enhancing activity of the presentinvention may be obtained from microorganisms of any genus. For purposesof the present invention, the term “obtained from” as used herein inconnection with a given source shall mean that the polypeptide encodedby a polynucleotide is produced by the source or by a strain in whichthe polynucleotide from the source has been inserted. In one aspect, thepolypeptide obtained from a given source is secreted extracellularly.

The polypeptide may be a bacterial polypeptide. For example, thepolypeptide may be a gram-positive bacterial polypeptide such as aBacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, orStreptomyces polypeptide having cellulolytic enhancing activity, or agram-negative bacterial polypeptide such as a Campylobacter, E. coli,Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria,Pseudomonas, Salmonella, or Ureaplasma polypeptide.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide.

In another aspect, the polypeptide is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus polypeptide.

In another aspect, the polypeptide is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans polypeptide.

The polypeptide may be a fungal polypeptide. For example, thepolypeptide may be a yeast polypeptide such as a Candida, Kluyveromyces,Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; ora filamentous fungal polypeptide such as an Acremonium, Agaricus,Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis,Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis,Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia,Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex,Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide.

In another aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis polypeptide.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium inops,Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporiummerdarium, Chrysosporium pannicola, Chrysosporium queenslandicum,Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa,Thielavia spededonium, Thielavia subthermophila, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride polypeptide.

In another aspect, the polypeptide is a Thielavia terrestris polypeptidehaving cellulolytic enhancing activity. In another aspect, thepolypeptide is a Thielavia terrestris NRRL 8126 polypeptide havingcellulolytic enhancing activity, e.g., the polypeptide comprising themature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) using the above-mentioned probes. Techniques for isolatingmicroorganisms from natural habitats are well known in the art. Thepolynucleotide encoding the polypeptide may then be obtained bysimilarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding apolypeptide has been detected with the probe(s), the polynucleotide canbe isolated or cloned by utilizing techniques that are well known tothose of ordinary skill in the art (see, e.g., Sambrook et al., 1989,supra).

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides from such genomic DNA can be effected, e.g., by usingthe well known polymerase chain reaction (PCR) or antibody screening ofexpression libraries to detect cloned DNA fragments with sharedstructural features. See, e.g., Innis et al., 1990, PCR: A Guide toMethods and Application, Academic Press, New York. Other nucleic acidamplification procedures such as ligase chain reaction (LCR), ligationactivated transcription (LAT) and polynucleotide-based amplification(NASBA) may be used. The polynucleotides may be cloned from a strain ofThielavia terrestris, or a related organism and thus, for example, maybe an allelic or species variant of the polypeptide encoding region ofthe polynucleotide.

The present invention also relates to isolated polynucleotides having asequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 5, or SEQ ID NO: 11 of at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100%; the mature polypeptide of SEQID NO: 3 of at least 65%, e.g., at least 70%, at least 75%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%;the mature polypeptide of SEQ ID NO: 17 of at least 70%, e.g., at least75%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%; the mature polypeptide of SEQ ID NO: 9, SEQ ID NO: 15, orSEQ ID NO: 21 of at least 75%, e.g., at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%; the mature polypeptideof SEQ ID NO: 7 of at least 80%, e.g., at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%; the mature polypeptide of SEQ ID NO:13 of at least 85%, e.g., at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%; or the mature polypeptide of SEQ ID NO: 19 at least90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%, which encode a polypeptide having cellulolytic enhancing activity.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., variantsthat differ in specific activity, thermostability, pH optimum, or thelike. The variant may be constructed on the basis of the polynucleotidepresented as the mature polypeptide coding sequence of SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO:21, or the cDNA sequences thereof, e.g., a subsequence thereof, and/orby introduction of nucleotide substitutions that do not result in achange in the amino acid sequence of the polypeptide, but whichcorrespond to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionsthat may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

The present invention also relates to isolated polynucleotides encodingpolypeptides of the present invention, which hybridize undermedium-high, high, or very high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 11, or SEQ ID NO: 17, (ii) the cDNA sequence containedin the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 11, or SEQ ID NO: 17, or (iii) the full-lengthcomplementary strand of (i) or (ii); high or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 21, (ii)the cDNA sequence contained in the mature polypeptide coding sequence ofSEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO:21, or (iii) the full-length complementary strand of (i) or (ii); orvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 19, (ii) the cDNA sequence contained in themature polypeptide coding sequence of SEQ ID NO: 19, or (iii) thefull-length complementary strand of (i) or (ii); or allelic variants andsubsequences thereof (Sambrook et al., 1989, supra), as defined herein.

In one aspect, the polynucleotide comprises or consists of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, or SEQ IDNO: 21, or the mature polypeptide coding sequences thereof, or asubsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, or SEQ ID NO: 21 that encodes a fragment of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, orSEQ ID NO: 22, respectively, having cellulolytic enhancing activity,such as the polynucleotide of nucleotides 52 to 875 of SEQ ID NO: 1,nucleotides 58 to 1250 of SEQ ID NO: 3, nucleotides 52 to 795 of SEQ IDNO: 5, nucleotides 58 to 974 of SEQ ID NO: 7, nucleotides 64 to 1104 ofSEQ ID NO: 9, nucleotides 73 to 990 of SEQ ID NO: 11, nucleotides 49 to1218 of SEQ ID NO: 13, nucleotides 55 to 930 of SEQ ID NO: 15,nucleotides 67 to 1581 of SEQ ID NO: 17, nucleotides 49 to 865 of SEQ IDNO: 19, or nucleotides 58 to 1065 of SEQ ID NO: 21.

In another aspect, the polynucleotide comprises or consists of SEQ IDNO: 1 or the mature polypeptide coding sequence thereof, which iscontained in plasmid pSMai216 which is contained in E. coli NRRLB-50301, wherein the polynucleotide sequence encodes a polypeptidehaving cellulolytic enhancing activity. In another aspect, thepolynucleotide comprises or consists of SEQ ID NO: 3 or the maturepolypeptide coding sequence thereof, which is contained in plasmidpSMAi217 which is contained in E. coli NRRL B-50302, wherein thepolynucleotide sequence encodes a polypeptide having cellulolyticenhancing activity. In another aspect, the polynucleotide comprises orconsists of SEQ ID NO: 5 or the mature polypeptide coding sequencethereof, which is contained in plasmid pSMai218 which is contained in E.coli NRRL B-50303, wherein the polynucleotide sequence encodes apolypeptide having cellulolytic enhancing activity. In another aspect,the polynucleotide comprises or consists of SEQ ID NO: 7 or the maturepolypeptide coding sequence thereof, which is contained in plasmidpSMai213 which is contained in E. coli NRRL B-50300, wherein thepolynucleotide sequence encodes a polypeptide having cellulolyticenhancing activity. In another aspect, the polynucleotide comprises orconsists of SEQ ID NO: 9 or the mature polypeptide coding sequencethereof, which is contained in plasmid pAG68 which is contained in E.coli NRRL B-50320, wherein the polynucleotide sequence encodes apolypeptide having cellulolytic enhancing activity. In another aspect,the polynucleotide comprises or consists of SEQ ID NO: 11 or the maturepolypeptide coding sequence thereof, which is contained in plasmid pAG69which is contained in E. coli NRRL B-50321, wherein the polynucleotidesequence encodes a polypeptide having cellulolytic enhancing activity.In another aspect, the polynucleotide comprises or consists of SEQ IDNO: 13 or the mature polypeptide coding sequence thereof, which iscontained in plasmid pAG75 which is contained in E. coli NRRL B-50322,wherein the polynucleotide sequence encodes a polypeptide havingcellulolytic enhancing activity. In another aspect, the polynucleotidecomprises or consists of SEQ ID NO: 15 or the mature polypeptide codingsequence thereof, which is contained in plasmid pAG76 which is containedin E. coli NRRL B-50323, wherein the polynucleotide sequence encodes apolypeptide having cellulolytic enhancing activity. In another aspect,the polynucleotide comprises or consists of SEQ ID NO: 17 or the maturepolypeptide coding sequence thereof, which is contained in plasmid pAG77which is contained in E. coli NRRL B-50324, wherein the polynucleotidesequence encodes a polypeptide having cellulolytic enhancing activity.In another aspect, the polynucleotide comprises or consists of SEQ IDNO: 19 or the mature polypeptide coding sequence thereof, which iscontained in plasmid pAG78 which is contained in E. coli NRRL B-50325,wherein the polynucleotide sequence encodes a polypeptide havingcellulolytic enhancing activity. In another aspect, the polynucleotidecomprises or consists of SEQ ID NO: 21 or the mature polypeptide codingsequence thereof, which is contained in plasmid pAG79 which is containedin E. coli NRRL B-50326, wherein the polynucleotide sequence encodes apolypeptide having cellulolytic enhancing activity.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or more(several) control sequences that direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences.

A polynucleotide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide priorto its insertion into a vector may be desirable or necessary dependingon the expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter sequence, a polynucleotide thatis recognized by a host cell for expression of a polynucleotide encodinga polypeptide of the present invention. The promoter sequence containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, E. colilac operon, Streptomyces coelicolor agarase gene (dagA), and prokaryoticbeta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci.USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983,Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are describedin “Useful proteins from recombinant bacteria” in Gilbert et al., 1980,Scientific American, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from a gene encoding a neutralalpha-amylase in Aspergilli in which the untranslated leader has beenreplaced by an untranslated leader from a gene encoding triose phosphateisomerase in Aspergilli; non-limiting examples include modifiedpromoters from the gene encoding neutral alpha-amylase in Aspergillusniger in which the untranslated leader has been replaced by anuntranslated leader from the gene encoding triose phosphate isomerase inAspergillus nidulans or Aspergillus oryzae); and mutant, truncated, andhybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, which is recognized by a host cell to terminate transcription.The terminator sequence is operably linked to the 3′-terminus of thepolynucleotide encoding the polypeptide. Any terminator that isfunctional in the host cell of choice may be used in the presentinvention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, whentranscribed is a nontranslated region of an mRNA that is important fortranslation by the host cell. The leader sequence is operably linked tothe 5′-terminus of the polynucleotide encoding the polypeptide. Anyleader sequence that is functional in the host cell of choice may beused.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell of choice may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. The foreign signal peptide coding sequence may be requiredwhere the coding sequence does not naturally contain a signal peptidecoding sequence. Alternatively, the foreign signal peptide codingsequence may simply replace the natural signal peptide coding sequencein order to enhance secretion of the polypeptide. However, any signalpeptide coding sequence that directs the expressed polypeptide into thesecretory pathway of a host cell of choice may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present at theN-terminus of a polypeptide, the propeptide sequence is positioned nextto the N-terminus of a polypeptide and the signal peptide sequence ispositioned next to the N-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those that causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the Aspergillus niger glucoamylasepromoter, Aspergillus oryzae TAKA alpha-amylase promoter, andAspergillus oryzae glucoamylase promoter may be used. Other examples ofregulatory sequences are those that allow for gene amplification. Ineukaryotic systems, these regulatory sequences include the dihydrofolatereductase gene that is amplified in the presence of methotrexate, andthe metallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the polypeptide would be operablylinked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more (several) convenientrestriction sites to allow for insertion or substitution of thepolynucleotide encoding the polypeptide at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the sequence into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more (several) selectable markersthat permit easy selection of transformed, transfected, transduced, orthe like cells. A selectable marker is a gene the product of whichprovides for biocide or viral resistance, resistance to heavy metals,prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAM111permitting replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or more(several) control sequences that direct the production of a polypeptideof the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any gram-positive or gram-negativebacterium. Gram-positive bacteria include, but not limited to, Bacillus,Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus,Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces.Gram-negative bacteria include, but not limited to, Campylobacter, E.coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter,Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Mol. Gen. Genet. 168: 111-115), by using competent cells (see, e.g.,Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), by electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or byconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may, forinstance, be effected by protoplast transformation (see, e.g., Hanahan,1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Doweretal., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNAinto a Streptomyces cell may, for instance, be effected by protoplasttransformation and electroporation (see, e.g., Gong etal., 2004, FoliaMicrobiol. (Praha) 49: 399-405), by conjugation (see, e.g., Mazodier etal., 1989, J. Bacteriol. 171: 3583-3585), or by transduction (see, e.g.,Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). Theintroduction of DNA into a Pseudomonas cell may, for instance, beeffected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol.Methods 64: 391-397) or by conjugation (see, e.g., Pinedo and Smets,2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA intoa Streptococcus cell may, for instance, be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), by protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), by electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or by conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (asdefined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary ofThe Fungi, 8th edition, 1995, CAB International, University Press,Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al.,1995, supra, page 171) and all mitosporic fungi (Hawksworth et al.,1995, supra).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, F. A., Passmore, S. M., andDavenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9,1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phiebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In a preferred aspect, the cell is of the genus Thielavia. In a morepreferred aspect, the cell is Thielavia terrestris. In a most preferredaspect, the cell is Thielavia terrestris NRRL 8126.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods well known in the art. Forexample, the cell may be cultivated by shake flask cultivation, andsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe polypeptide to be expressed and/or isolated. The cultivation takesplace in a suitable nutrient medium comprising carbon and nitrogensources and inorganic salts, using procedures known in the art. Suitablemedia are available from commercial suppliers or may be preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection). If the polypeptide is secreted into thenutrient medium, the polypeptide can be recovered directly from themedium. If the polypeptide is not secreted, it can be recovered fromcell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, centrifugation,filtration, extraction, spray-drying, evaporation, or precipitation.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989) to obtainsubstantially pure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising an isolatedpolynucleotide of the present invention so as to express and produce thepolypeptide in recoverable quantities. The polypeptide may be recoveredfrom the plant or plant part. Alternatively, the plant or plant partcontaining the polypeptide may be used as such for improving the qualityof a food or feed, e.g., improving nutritional value, palatability, andrheological properties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells. The transgenic plant orplant cell expressing a polypeptide may be constructed in accordancewith methods known in the art. In short, the plant or plant cell isconstructed by incorporating one or more (several) expression constructsencoding a polypeptide into the plant host genome or chloroplast genomeand propagating the resulting modified plant or plant cell into atransgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide operably linked withappropriate regulatory sequences required for expression of thepolynucleotide in the plant or plant part of choice. Furthermore, theexpression construct may comprise a selectable marker useful foridentifying host cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide may be constitutive or inducible, or may be developmental,stage or tissue specific, and the gene product may be targeted to aspecific tissue or plant part such as seeds or leaves. Regulatorysequences are, for example, described by Tague et al., 1988, PlantPhysiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay inducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide in the plant. For instance, the promoterenhancer element may be an intron that is placed between the promoterand the polynucleotide encoding a polypeptide. For instance, Xu et al.,1993, supra, disclose the use of the first intron of the rice actin 1gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Mol. Biol. 19: 15-38) and can alsobe used for transforming monocots, although other transformation methodsare often used for these plants. Presently, the method of choice forgenerating transgenic monocots is particle bombardment (microscopic goldor tungsten particles coated with the transforming DNA) of embryoniccalli or developing embryos (Christou, 1992, Plant J. 2: 275-281;Shimamoto, 1994, Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992,Bio/Technology 10: 667-674). An alternative method for transformation ofmonocots is based on protoplast transformation as described by Omirullehet al., 1993, Plant Mol. Biol. 21: 415-428. Additional transformationmethods for use in accordance with the present disclosure include thosedescribed in U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which areherein incorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct prepared according to the present invention, transgenicplants may be made by crossing a plant having the construct to a secondplant lacking the construct. For example, a construct encoding apolypeptide can be introduced into a particular plant variety bycrossing, without the need for ever directly transforming a plant ofthat given variety. Therefore, the present invention encompasses notonly a plant directly regenerated from cells which have been transformedin accordance with the present invention, but also the progeny of suchplants. As used herein, progeny may refer to the offspring of anygeneration of a parent plant prepared in accordance with the presentinvention. Such progeny may include a DNA construct prepared inaccordance with the present invention, or a portion of a DNA constructprepared in accordance with the present invention. Crossing results inthe introduction of a transgene into a plant line by cross pollinating astarting line with a donor plant line. Non-limiting examples of suchsteps are further articulated in U.S. Pat. No. 7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a polypeptideof the present invention comprising: (a) cultivating a transgenic plantor a plant cell comprising a polynucleotide encoding the polypeptideunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Removal or Reduction of Cellulolytic Enhancing Activity

The present invention also relates to methods of producing a mutant of aparent cell, which comprises disrupting or deleting a polynucleotide, ora portion thereof, encoding a polypeptide of the present invention,which results in the mutant cell producing less of the polypeptide thanthe parent cell when cultivated under the same conditions.

The mutant cell may be constructed by reducing or eliminating expressionof the polynucleotide using methods well known in the art, for example,insertions, disruptions, replacements, or deletions. In a preferredaspect, the polynucleotide is inactivated. The polynucleotide to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required for theexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thepolynucleotide. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the polynucleotide may be performed bysubjecting the parent cell to mutagenesis and selecting for mutant cellsin which expression of the polynucleotide has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), 0-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the polynucleotide may be accomplishedby introduction, substitution, or removal of one or more (several)nucleotides in the gene or a regulatory element required for thetranscription or translation thereof. For example, nucleotides may beinserted or removed so as to result in the introduction of a stop codon,the removal of the start codon, or a change in the open reading frame.Such modification or inactivation may be accomplished by site-directedmutagenesis or PCR generated mutagenesis in accordance with methodsknown in the art. Although, in principle, the modification may beperformed in vivo, i.e., directly on the cell expressing thepolynucleotide to be modified, it is preferred that the modification beperformed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce expression of apolynucleotide is based on techniques of gene replacement, genedeletion, or gene disruption. For example, in the gene disruptionmethod, a nucleic acid sequence corresponding to the endogenouspolynucleotide is mutagenized in vitro to produce a defective nucleicacid sequence that is then transformed into the parent cell to produce adefective gene. By homologous recombination, the defective nucleic acidsequence replaces the endogenous polynucleotide. It may be desirablethat the defective polynucleotide also encodes a marker that may be usedfor selection of transformants in which the polynucleotide has beenmodified or destroyed. In a particularly preferred aspect, thepolynucleotide is disrupted with a selectable marker such as thosedescribed herein.

The present invention also relates to methods of inhibiting theexpression of a polypeptide having cellulolytic enhancing activity in acell, comprising administering to the cell or expressing in the cell adouble-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises asubsequence of a polynucleotide of the present invention. In a preferredaspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length.

The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA(miRNA). In a preferred aspect, the dsRNA is small interfering RNA(siRNAs) for inhibiting transcription. In another preferred aspect, thedsRNA is micro RNA (miRNAs) for inhibiting translation.

The present invention also relates to such double-stranded RNA (dsRNA)molecules, comprising a portion of the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,SEQ ID NO: 19, or SEQ ID NO: 21 for inhibiting expression of thepolypeptide in a cell. While the present invention is not limited by anyparticular mechanism of action, the dsRNA can enter a cell and cause thedegradation of a single-stranded RNA (ssRNA) of similar or identicalsequences, including endogenous mRNAs. When a cell is exposed to dsRNA,mRNA from the homologous gene is selectively degraded by a processcalled RNA interference (RNAi).

The dsRNAs of the present invention can be used in gene-silencing. Inone aspect, the invention provides methods to selectively degrade RNAusing a dsRNAi of the present invention. The process may be practiced invitro, ex vivo or in vivo. In one aspect, the dsRNA molecules can beused to generate a loss-of-function mutation in a cell, an organ or ananimal. Methods for making and using dsRNA molecules to selectivelydegrade RNA are well known in the art; see, for example, U.S. Pat. Nos.6,489,127; 6,506,559; 6,511,824; and 6,515,109.

The present invention further relates to a mutant cell of a parent cellthat comprises a disruption or deletion of a polynucleotide encoding thepolypeptide or a control sequence thereof or a silenced gene encodingthe polypeptide, which results in the mutant cell producing less of thepolypeptide or no polypeptide compared to the parent cell.

The polypeptide-deficient mutant cells are particularly useful as hostcells for the expression of native and heterologous polypeptides.Therefore, the present invention further relates to methods of producinga native or heterologous polypeptide, comprising: (a) cultivating themutant cell under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide. The term “heterologouspolypeptides” means polypeptides that are not native to the host cell,e.g., a variant of a native protein. The host cell may comprise morethan one copy of a polynucleotide encoding the native or heterologouspolypeptide.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallycellulolytic enhancing-free product are of particular interest in theproduction of eukaryotic polypeptides, in particular fungal proteinssuch as enzymes. The cellulolytic enhancing-deficient cells may also beused to express heterologous proteins of pharmaceutical interest such ashormones, growth factors, receptors, and the like. The term “eukaryoticpolypeptides” includes not only native polypeptides, but also thosepolypeptides, e.g., enzymes, which have been modified by amino acidsubstitutions, deletions or additions, or other such modifications toenhance activity, thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from cellulolytic enhancing activity that is producedby a method of the present invention.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that theendoglucanase activity of the composition has been increased, e.g., withan enrichment factor of at least 1.1.

The composition may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as one or more (several) enzymes selected from thegroup consisting of a cellulase, a hemicellulase, an expansin, anesterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Uses

The present invention is also directed to the following methods forusing the polypeptides having cellulolytic enhancing activity, orcompositions thereof.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity of the present invention. In oneaspect, the method further comprises recovering the degraded orconverted cellulosic material. Soluble products of degradation orconversion of the cellulosic material can be separated from theinsoluble cellulosic material using technology well known in the artsuch as, for example, centrifugation, filtration, and gravity settling.

The present invention also relates to methods of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity of the present invention; (b)fermenting the saccharified cellulosic material with one or more(several) fermenting microorganisms to produce the fermentation product;and (c) recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition in the presence of apolypeptide having cellulolytic enhancing activity of the presentinvention. In one aspect, the fermenting of the cellulosic materialproduces a fermentation product. In another aspect, the method furthercomprises recovering the fermentation product from the fermentation.

The methods of the present invention can be used to saccharify thecellulosic material to fermentable sugars and convert the fermentablesugars to many useful substances, e.g., fuel, potable ethanol, and/orfermentation products (e.g., acids, alcohols, ketones, gases, and thelike). The production of a desired fermentation product from thecellulosic material typically involves pretreatment, enzymatichydrolysis (saccharification), and fermentation.

The processing of the cellulosic material according to the presentinvention can be accomplished using processes conventional in the art.Moreover, the methods of the present invention can be implemented usingany conventional biomass processing apparatus configured to operate inaccordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and cofermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF);and direct microbial conversion (DMC). SHF uses separate process stepsto first enzymatically hydrolyze the cellulosic material to fermentablesugars, e.g., glucose, cellobiose, cellotriose, and pentose sugars, andthen ferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of the cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, DC,179-212). SSCF involves the cofermentation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog. 15: 817-827).HHF involves a separate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more(several) steps where the same organism is used to produce the enzymesfor conversion of the cellulosic material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd, L. R.,Weimer, P. J., van Zyl, W. H., and Pretorius, I. S., 2002, Microbialcellulose utilization: Fundamentals and biotechnology, Microbiol. Mol.Biol. Reviews 66: 506-577). It is understood herein that any methodknown in the art comprising pretreatment, enzymatic hydrolysis(saccharification), fermentation, or a combination thereof, can be usedin the practicing the methods of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Fernandade Castilhos Corazza, Flavio Faria de Moraes, Gisella Maria Zanin andIvo Neitzel, 2003, Optimal control in fed-batch reactor for thecellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov,A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysisof cellulose: 1. A mathematical model for a batch reactor process, Enz.Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee,J. M., 1983, Bioconversion of waste cellulose by using an attritionbioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensivestirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn,A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, 0. V., 1996,Enhancement of enzymatic cellulose hydrolysis using a novel type ofbioreactor with intensive stirring induced by electromagnetic field,Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor typesinclude: fluidized bed, upflow blanket, immobilized, and extruder typereactors for hydrolysis and/or fermentation.

Pretreatment. In practicing the methods of the present invention, anypretreatment process known in the art can be used to disrupt plant cellwall components of the cellulosic material (Chandra et al., 2007,Substrate pretreatment: The key to effective enzymatic hydrolysis oflignocellulosics? Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe andZacchi, 2007, Pretreatment of lignocellulosic materials for efficientbioethanol production, Adv. Biochem. Engin./Biotechnol. 108: 41-65;Hendriks and Zeeman, 2009, Pretreatments to enhance the digestibility oflignocellulosic biomass, Bioresource Technol. 100: 10-18; Mosier et al.,2005, Features of promising technologies for pretreatment oflignocellulosic biomass, Bioresource Technol. 96: 673-686; Taherzadehand Karimi, 2008, Pretreatment of lignocellulosic wastes to improveethanol and biogas production: A review, Int. J. of Mol. Sci. 9:1621-1651; Yang and Wyman, 2008, Pretreatment: the key to unlockinglow-cost cellulosic ethanol, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, pre-soaking, wetting, washing, and/or conditioning prior topretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, and gamma irradiationpretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment: In steam pretreatment, the cellulosic material isheated to disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. The cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably done at 140-230° C., more preferably 160-200° C., and mostpreferably 170-190° C., where the optimal temperature range depends onany addition of a chemical catalyst. Residence time for the steampretreatment is preferably 1-15 minutes, more preferably 3-12 minutes,and most preferably 4-10 minutes, where the optimal residence timedepends on temperature range and any addition of a chemical catalyst.Steam pretreatment allows for relatively high solids loadings, so thatthe cellulosic material is generally only moist during the pretreatment.The steam pretreatment is often combined with an explosive discharge ofthe material after the pretreatment, which is known as steam explosion,that is, rapid flashing to atmospheric pressure and turbulent flow ofthe material to increase the accessible surface area by fragmentation(Duff and Murray, 1996, Bioresource Technology 855: 1-33; Galbe andZacchi, 2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. PatentApplication No. 20020164730). During steam pretreatment, hemicelluloseacetyl groups are cleaved and the resulting acid autocatalyzes partialhydrolysis of the hemicellulose to monosaccharides and oligosaccharides.Lignin is removed to only a limited extent.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 3% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (Ballesteroset al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006,Enzyme Microb. Technol. 39: 756-762).

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Examples of suitable chemicalpretreatment processes include, for example, dilute acid pretreatment,lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX),ammonia percolation (APR), and organosolv pretreatments.

In dilute acid pretreatment, the cellulosic material is mixed withdilute acid, typically H2SO4, and water to form a slurry, heated bysteam to the desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs, e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors (Duff andMurray, 1996, supra; Schell et al., 2004, Bioresource TechnoL 91:179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to, limepretreatment, wet oxidation, ammonia percolation (APR), and ammoniafiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium carbonate, sodium hydroxide,or ammonia at low temperatures of 85-150° C. and residence times from 1hour to several days (Wyman et al., 2005, Bioresource TechnoL 96:1959-1966; Mosier et al., 2005, Bioresource TechnoL 96: 673-686). WO2006/110891, WO 2006/11899, WO 2006/11900, and WO 2006/110901 disclosepretreatment methods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource TechnoL 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. TechnoL Biotechnol. 81:1669-1677). The pretreatment is performed at preferably 1-40% drymatter, more preferably 2-30% dry matter, and most preferably 5-20% drymatter, and often the initial pH is increased by the addition of alkalisuch as sodium carbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion), can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves treating the cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-100°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96:2014-2018). AFEX pretreatment results in the depolymerization ofcellulose and partial hydrolysis of hemicellulose. Lignin-carbohydratecomplexes are cleaved.

Organosolv pretreatment delignifies the cellulosic material byextraction using aqueous ethanol (40-60% ethanol) at 160-200° C. for30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan etal., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl.Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose isremoved.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as anacid treatment, and more preferably as a continuous dilute and/or mildacid treatment. The acid is typically sulfuric acid, but other acids canalso be used, such as acetic acid, citric acid, nitric acid, phosphoricacid, tartaric acid, succinic acid, hydrogen chloride, or mixturesthereof. Mild acid treatment is conducted in the pH range of preferably1-5, more preferably 1-4, and most preferably 1-3. In one aspect, theacid concentration is in the range from preferably 0.01 to 20 wt % acid,more preferably 0.05 to 10 wt % acid, even more preferably 0.1 to 5 wt %acid, and most preferably 0.2 to 2.0 wt % acid. The acid is contactedwith the cellulosic material and held at a temperature in the range ofpreferably 160-220° C., and more preferably 165-195° C., for periodsranging from seconds to minutes to, e.g., 1 second to 60 minutes.

In another aspect, pretreatment is carried out as an ammonia fiberexplosion step (AFEX pretreatment step).

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the cellulosic material is present duringpretreatment in amounts preferably between 10-80 wt %, more preferablybetween 20-70 wt %, and most preferably between 30-60 wt %, such asaround 50 wt %. The pretreated cellulosic material can be unwashed orwashed using any method known in the art, e.g., washed with water.

Mechanical Pretreatment: The term “mechanical pretreatment” refers tovarious types of grinding or milling (e.g., dry milling, wet milling, orvibratory ball milling).

Physical Pretreatment: The term “physical pretreatment” refers to anypretreatment that promotes the separation and/or release of cellulose,hemicellulose, and/or lignin from the cellulosic material. For example,physical pretreatment can involve irradiation (e.g., microwaveirradiation), steaming/steam explosion, hydrothermolysis, andcombinations thereof.

Physical pretreatment can involve high pressure and/or high temperature(steam explosion). In one aspect, high pressure means pressure in therange of preferably about 300 to about 600 psi, more preferably about350 to about 550 psi, and most preferably about 400 to about 500 psi,such as around 450 psi. In another aspect, high temperature meanstemperatures in the range of about 100 to about 300° C., preferablyabout 140 to about 235° C. In a preferred aspect, mechanicalpretreatment is performed in a batch-process, steam gun hydrolyzersystem that uses high pressure and high temperature as defined above,e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden.

Combined Physical and Chemical Pretreatment: The cellulosic material canbe pretreated both physically and chemically. For instance, thepretreatment step can involve dilute or mild acid treatment and hightemperature and/or pressure treatment. The physical and chemicalpretreatments can be carried out sequentially or simultaneously, asdesired. A mechanical pretreatment can also be included.

Accordingly, in a preferred aspect, the cellulosic material is subjectedto mechanical, chemical, or physical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms (see, for example, Hsu, T.-A., 1996,Pretreatment of biomass, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, DC,179-212; Ghosh and Singh, 1993, Physicochemical and biologicaltreatments for enzymatic/microbial conversion of cellulosic biomass,Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreatinglignocellulosic biomass: a review, in Enzymatic Conversion of Biomassfor Fuels Production, Himmel, M. E., Baker, J. O., and Overend, R. P.,eds., ACS Symposium Series 566, American Chemical Society, Washington,DC, chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999,Ethanol production from renewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996,Fermentation of lignocellulosic hydrolysates for ethanol production,Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990,Production of ethanol from lignocellulosic materials: State of the art,Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification. In the hydrolysis step, also known assaccharification, the cellulosic material, e.g., pretreated, ishydrolyzed to break down cellulose and alternatively also hemicelluloseto fermentable sugars, such as glucose, cellobiose, xylose, xylulose,arabinose, mannose, galactose, and/or soluble oligosaccharides. Thehydrolysis is performed enzymatically by an enzyme composition in thepresence of a polypeptide having cellulolytic enhancing activity of thepresent invention. The enzymes of the compositions can be addedsequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In one aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme(s), i.e., optimal forthe enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the cellulosic material is fed gradually to,for example, an enzyme containing hydrolysis solution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 96 hours, more preferably about 16 to about 72 hours,and most preferably about 24 to about 48 hours. The temperature is inthe range of preferably about 25° C. to about 70° C., more preferablyabout 30° C. to about 65° C., and more preferably about 40° C. to 60°C., in particular about 50° C. The pH is in the range of preferablyabout 3 to about 8, more preferably about 3.5 to about 7, and mostpreferably about 4 to about 6, in particular about pH 5. The dry solidscontent is in the range of preferably about 5 to about 50 wt %, morepreferably about 10 to about 40 wt %, and most preferably about 20 toabout 30 wt %.

The enzyme compositions can comprise any protein that is useful indegrading or converting the cellulosic material.

In one aspect, the enzyme composition comprises or further comprises oneor more (several) proteins selected from the group consisting of acellulase, a polypeptide having cellulolytic enhancing activity, ahemicellulase, an expansin, an esterase, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin. Inanother aspect, the cellulase is preferably one or more (several)enzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase. In another aspect, thehemicellulase is preferably one or more (several) enzymes selected fromthe group consisting of an acetylmannan esterase, an acetyxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase.

In another aspect, the enzyme composition comprises one or more(several) cellulolytic enzymes. In another aspect, the enzymecomposition comprises or further comprises one or more (several) hemicellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (several) cellulolytic enzymes and one or more(several) hemicellulolytic enzymes. In another aspect, the enzymecomposition comprises one or more (several) enzymes selected from thegroup of cellulolytic enzymes and hemicellulolytic enzymes. In anotheraspect, the enzyme composition comprises an endoglucanase. In anotheraspect, the enzyme composition comprises a cellobiohydrolase. In anotheraspect, the enzyme composition comprises a beta-glucosidase. In anotheraspect, the enzyme composition comprises a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises an endoglucanase and a cellobiohydrolase. Inanother aspect, the enzyme composition comprises an endoglucanase and abeta-glucosidase. In another aspect, the enzyme composition comprises acellobiohydrolase and a beta-glucosidase. In another aspect, the enzymecomposition comprises an endoglucanase, a cellobiohydrolase, and abeta-glucosidase. In another aspect, the enzyme composition comprises anendoglucanase, a cellobiohydrolase, a beta-glucosidase, and apolypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises an acetylmannan esterase. In anotheraspect, the enzyme composition comprises an acetyxylan esterase. Inanother aspect, the enzyme composition comprises an arabinanase (e.g.,alpha-L-arabinanase). In another aspect, the enzyme compositioncomprises an arabinofuranosidase (e.g., alpha-L-arabinofuranosidase). Inanother aspect, the enzyme composition comprises a coumaric acidesterase. In another aspect, the enzyme composition comprises a feruloylesterase. In another aspect, the enzyme composition comprises agalactosidase (e.g., alpha-galactosidase and/or beta-galactosidase). Inanother aspect, the enzyme composition comprises a glucuronidase (e.g.,alpha-D-glucuronidase). In another aspect, the enzyme compositioncomprises a glucuronoyl esterase. In another aspect, the enzymecomposition comprises a mannanase. In another aspect, the enzymecomposition comprises a mannosidase (e.g., beta-mannosidase). In anotheraspect, the enzyme composition comprises a xylanase. In a preferredaspect, the xylanase is a Family 10 xylanase. In another aspect, theenzyme composition comprises a xylosidase (e.g., beta-xylosidase). Inanother aspect, the enzyme composition comprises an expansin. In anotheraspect, the enzyme composition comprises an esterase. In another aspect,the enzyme composition comprises a laccase. In another aspect, theenzyme composition comprises a ligninolytic enzyme. In a preferredaspect, the ligninolytic enzyme is a manganese peroxidase. In anotherpreferred aspect, the ligninolytic enzyme is a lignin peroxidase. Inanother preferred aspect, the ligninolytic enzyme is a H₂O₂-producingenzyme. In another aspect, the enzyme composition comprises a pectinase.In another aspect, the enzyme composition comprises a peroxidase. Inanother aspect, the enzyme composition comprises a protease. In anotheraspect, the enzyme composition comprises a swollenin.

In the methods of the present invention, the enzyme(s) can be addedprior to or during fermentation, e.g., during saccharification or duringor after propagation of the fermenting microorganism(s).

One or more (several) components of the enzyme composition may bewild-type proteins, recombinant proteins, or a combination of wild-typeproteins and recombinant proteins. For example, one or more (several)components may be native proteins of a cell, which is used as a hostcell to express recombinantly one or more (several) other components ofthe enzyme composition. One or more (several) components of the enzymecomposition may be produced as monocomponents, which are then combinedto form the enzyme composition. The enzyme composition may be acombination of multicomponent and monocomponent protein preparations.

The enzymes used in the methods of the present invention may be in anyform suitable for use, such as, for example, a crude fermentation brothwith or without cells removed, a cell lysate with or without cellulardebris, a semi-purified or purified enzyme preparation, or a host cellas a source of the enzymes. The enzyme composition may be a dry powderor granulate, a non-dusting granulate, a liquid, a stabilized liquid, ora stabilized protected enzyme. Liquid enzyme preparations may, forinstance, be stabilized by adding stabilizers such as a sugar, a sugaralcohol or another polyol, and/or lactic acid or another organic acidaccording to established processes.

The optimum amounts of the enzymes and a polypeptide having cellulolyticenhancing activity depend on several factors including, but not limitedto, the mixture of component cellulolytic enzymes, the cellulosicmaterial, the concentration of cellulosic material, the pretreatment(s)of the cellulosic material, temperature, time, pH, and inclusion offermenting organism (e.g., yeast for Simultaneous Saccharification andFermentation).

In a preferred aspect, an effective amount of cellulolytic enzyme to thecellulosic material is about 0.5 to about 50 mg, preferably about 0.5 toabout 40 mg, more preferably about 0.5 to about 25 mg, more preferablyabout 0.75 to about 20 mg, more preferably about 0.75 to about 15 mg,even more preferably about 0.5 to about 10 mg, and most preferably about2.5 to about 10 mg per g of the cellulosic material.

In another preferred aspect, an effective amount of a polypeptide havingcellulolytic enhancing activity to the cellulosic material is about 0.01to about 50.0 mg, preferably about 0.01 to about 40 mg, more preferablyabout 0.01 to about 30 mg, more preferably about 0.01 to about 20 mg,more preferably about 0.01 to about 10 mg, more preferably about 0.01 toabout 5 mg, more preferably about 0.025 to about 1.5 mg, more preferablyabout 0.05 to about 1.25 mg, more preferably about 0.075 to about 1.25mg, more preferably about 0.1 to about 1.25 mg, even more preferablyabout 0.15 to about 1.25 mg, and most preferably about 0.25 to about 1.0mg per g of the cellulosic material.

In another preferred aspect, an effective amount of a polypeptide havingcellulolytic enhancing activity to cellulolytic enzyme is about 0.005 toabout 1.0 g, preferably about 0.01 to about 1.0 g, more preferably about0.15 to about 0.75 g, more preferably about 0.15 to about 0.5 g, morepreferably about 0.1 to about 0.5 g, even more preferably about 0.1 toabout 0.25 g, and most preferably about 0.05 to about 0.2 g per g ofcellulolytic enzyme.

The polypeptides having cellulolytic enzyme activity or hemicellulolyticenzyme activity as well as other proteins/polypeptides useful in thedegradation of the cellulosic material, e.g., polypeptides havingcellulolytic enhancing activity (hereinafter “polypeptides having enzymeactivity”) can be derived or obtained from any suitable origin,including, bacterial, fungal, yeast, plant, or mammalian origin. Theterm “obtained” means herein that the enzyme may have been isolated froman organism that naturally produces the enzyme as a native enzyme. Theterm “obtained” also means herein that the enzyme may have been producedrecombinantly in a host organism employing methods described herein,wherein the recombinantly produced enzyme is either native or foreign tothe host organism or has a modified amino acid sequence, e.g., havingone or more (several) amino acids that are deleted, inserted and/orsubstituted, i.e., a recombinantly produced enzyme that is a mutantand/or a fragment of a native amino acid sequence or an enzyme producedby nucleic acid shuffling processes known in the art. Encompassed withinthe meaning of a native enzyme are natural variants and within themeaning of a foreign enzyme are variants obtained recombinantly, such asby site-directed mutagenesis or shuffling.

A polypeptide having enzyme activity may be a bacterial polypeptide. Forexample, the polypeptide may be a gram positive bacterial polypeptidesuch as a Bacillus, Streptococcus, Streptomyces, Staphylococcus,Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, orOceanobacillus polypeptide having enzyme activity, or a Gram negativebacterial polypeptide such as an E. coli, Pseudomonas, Salmonella,Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter,Neisseria, or Ureaplasma polypeptide having enzyme activity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having enzyme activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide having enzymeactivity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide having enzymeactivity.

The polypeptide having enzyme activity may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide having enzyme activity; or more preferably a filamentousfungal polypeptide such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Dipodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, lrpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having enzymeactivity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having enzymeactivity.

In another preferred aspect, the polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicolainsolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum,Penicillium purpurogenum, Phanerochaete chrysosporium, Thielaviaachromatica, Thielavia albomyces, Thielavia albopilosa, Thielaviaaustraleinsis, Thielavia fimeti, Thielavia microspora, Thielaviaovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa,Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei ,Trichoderma viride, or Trichophaea saccata polypeptide having enzymeactivity.

Chemically modified or protein engineered mutants of polypeptides havingenzyme activity may also be used.

One or more (several) components of the enzyme composition may be arecombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host is preferably a heterologous host (enzyme isforeign to host), but the host may under certain conditions also be ahomologous host (enzyme is native to host). Monocomponent cellulolyticproteins may also be prepared by purifying such a protein from afermentation broth.

In one aspect, the one or more (several) cellulolytic enzymes comprise acommercial cellulolytic enzyme preparation. Examples of commercialcellulolytic enzyme preparations suitable for use in the presentinvention include, for example, CELLIC™ CTec (Novozymes A/S),CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188 (Novozymes A/S), CELLUZYME™(Novozymes A/S), CEREFLO™ (Novozymes A/S), and ULTRAFLO™ (NovozymesA/S), ACCELERASE™ (Genencor Int.), LAMINEX™ (Genencor Int.), SPEZYME™ CP(Genencor Int.), ROHAMENT™ 7069 W (Rohm GmbH), FIBREZYME® LDI (DyadicInternational, Inc.), FIBREZYME® LBR (Dyadic International, Inc.), orVISCOSTAR® 150L (Dyadic International, Inc.). The cellulase enzymes areadded in amounts effective from about 0.001 to about 5.0 wt % of solids,more preferably from about 0.025 to about 4.0 wt % of solids, and mostpreferably from about 0.005 to about 2.0 wt % of solids.

Examples of bacterial endoglucanases that can be used in the methods ofthe present invention, include, but are not limited to, an Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263; Trichodermareesei Cel7B endoglucanase I; GENBANK™ accession no. M15665);Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene63:11-22; Trichoderma reesei Cel5A endoglucanase II; GENBANK™ accessionno. M19373); Trichoderma reesei endoglucanase Ill (Okada et al., 1988,Appl. Environ. Microbiol. 64: 555-563; GENBANK™ accession no. AB003694);Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, MolecularMicrobiology 13: 219-228; GENBANK™ accession no. Z33381); Aspergillusaculeatus endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18:5884); Aspergillus kawachii endoglucanase (Sakamoto et al., 1995,Current Genetics 27: 435-439); Erwinia carotovara endoglucanase(Saarilahti et al., 1990, Gene 90: 9-14); Fusarium oxysporumendoglucanase (GENBANK™ accession no. L29381); Humicola grisea var.thermoidea endoglucanase (GENBANK™ accession no. AB003107); Melanocarpusalbomyces endoglucanase (GENBANK™ accession no. MAL515703); Neurosporacrassa endoglucanase (GENBANK™ accession no. XM_324477); Humicolainsolens endoglucanase V; Myceliophthora thermophila CBS 117.65endoglucanase; basidiomycete CBS 495.95 endoglucanase; basidiomycete CBS494.95 endoglucanase; Thielavia terrestris NRRL 8126 CEL6Bendoglucanase; Thielavia terrestris NRRL 8126 CEL6C endoglucanase;Thielavia terrestris NRRL 8126 CEL7C endoglucanase; Thielavia terrestrisNRRL 8126 CEL7E endoglucanase; Thielavia terrestris NRRL 8126 CEL7Fendoglucanase; Cladorrhinum foecundissimum ATCC 62373 CEL7Aendoglucanase; and Trichoderma reesei strain No. VTT-D-80133endoglucanase (GENBANK™ accession no. M15665).

Examples of cellobiohydrolases useful in the present invention include,but are not limited to, Trichoderma reesei cellobiohydrolase I;Trichoderma reesei cellobiohydrolase II; Humicola insolenscellobiohydrolase I; Myceliophthora thermophila cellobiohydrolase II;Thielavia terrestris cellobiohydrolase II (CEL6A); Chaetomiumthermophilum cellobiohydrolase I; and Chaetomium thermophilumcellobiohydrolase II.

Examples of beta-glucosidases useful in the present invention include,but are not limited to, Aspergillus oryzae beta-glucosidase; Aspergillusfumigatus beta-glucosidase; Penicillium brasilianum IBT 20888beta-glucosidase; Aspergillus niger beta-glucosidase; and Aspergillusaculeatus beta-glucosidase.

The Aspergillus oryzae polypeptide having beta-glucosidase activity canbe obtained according to WO 2002/095014. The Aspergillus fumigatuspolypeptide having beta-glucosidase activity can be obtained accordingto WO 2005/047499. The Penicillium brasilianum polypeptide havingbeta-glucosidase activity can be obtained according to WO 2007/019442.The Aspergillus niger polypeptide having beta-glucosidase activity canbe obtained according to Dan et al., 2000, J. Biol. Chem. 275:4973-4980. The Aspergillus aculeatus polypeptide having beta-glucosidaseactivity can be obtained according to Kawaguchi et al., 1996, Gene 173:287-288.

The beta-glucosidase may be a fusion protein. In one aspect, thebeta-glucosidase is the Aspergillus oryzae beta-glucosidase variant BGfusion protein or the Aspergillus oryzae beta-glucosidase fusion proteinobtained according to WO 2008/057637.

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidasesare disclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat B., 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

Other cellulolytic enzymes that may be used in the present invention aredescribed in EP 495,257, EP 531,315, EP 531,372, WO 89/09259, WO94/07998, WO 95/24471, WO 96/11262, WO 96/29397, WO 96/034108, WO97/14804, WO 98/08940, WO 98/012307, WO 98/13465, WO 98/015619, WO98/015633, WO 98/028411, WO 99/06574, WO 99/10481, WO 99/025846, WO99/025847, WO 99/031255, WO 2000/009707, WO 2002/050245, WO2002/0076792, WO 2002/101078, WO 2003/027306, WO 2003/052054, WO2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/052118, WO2004/016760, WO 2004/043980, WO 2004/048592, WO 2005/001065, WO2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO2006/117432, WO 2007/071818, WO 2007/071820, WO 2008/008070, WO2008/008793, U.S. Pat. Nos. 4,435,307, 5,457,046, 5,648,263, 5,686,593,5,691,178, 5,763,254, and 5,776,757.

Examples of polypeptides having cellulolytic enhancing activity usefulin the methods of the present invention include, but are not limited to,polypeptides having cellulolytic enhancing activity from Thielaviaterrestris (WO 2005/074647); polypeptides having cellulolytic enhancingactivity from Thermoascus aurantiacus (WO 2005/074656); polypeptideshaving cellulolytic enhancing activity from Trichoderma reesei (WO2007/089290); and polypeptides having cellulolytic enhancing activityfrom Myceliophthora thermophila (WO 2009/085935; WO 2009/085859; WO2009/085864; WO 2009/085868).

In one aspect, the one or more (several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes A/S),CELLIC™ HTec (Novozymes A/S), VISCOZYME® (Novozymes A/S), ULTRAFLO®(Novozymes A/S), PULPZYME® HC (Novozymes A/S), MULTIFECT® Xylanase(Genencor), ECOPULP® TX-200A (AB Enzymes), HSP 6000 Xylanase (DSM),DEPOL™ 333P (Biocatalysts Limit, Wales, UK), DEPOL™ 740L. (BiocatalystsLimit, Wales, UK), and DEPOL™ 762P (Biocatalysts Limit, Wales, UK).

Examples of xylanases useful in the methods of the present inventioninclude, but are not limited to, Aspergillus aculeatus xylanase(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus xylanases (WO2006/078256), and Thielavia terrestris NRRL 8126 xylanases (WO2009/079210).

Examples of beta-xylosidases useful in the methods of the presentinvention include, but are not limited to, Trichoderma reeseibeta-xylosidase (UniProtKB/TrEMBL accession number Q92458), Talaromycesemersonii (SwissProt accession number Q8X212), and Neurospora crassa(SwissProt accession number Q7SOW4).

Examples of acetylxylan esterases useful in the methods of the presentinvention include, but are not limited to, Hypocrea jecorina acetylxylanesterase (WO 2005/001036), Neurospora crassa acetylxylan esterase(UniProt accession number q7s259), Thielavia terrestris NRRL 8126acetylxylan esterase (WO 2009/042846), Chaetomium globosum acetylxylanesterase (Uniprot accession number Q2GWX4), Chaetomium gracileacetylxylan esterase (GeneSeqP accession number AAB82124), Phaeosphaerianodorum acetylxylan esterase (Uniprot accession number Q0UHJ1), andHumicola insolens DSM 1800 acetylxylan esterase (WO 2009/073709).

Examples of ferulic acid esterases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800feruloyl esterase (WO 2009/076122), Neurospora crassa feruloyl esterase(UniProt accession number Q9HGR3), and Neosartorya fischeri feruloylesterase (UniProt Accession number Al D9T4).

Examples of arabinofuranosidases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800arabinofuranosidase (WO 2009/073383) and Aspergillus nigerarabinofuranosidase (GeneSeqP accession number AAR94170).

Examples of alpha-glucuronidases useful in the methods of the presentinvention include, but are not limited to, Aspergillus clavatusalpha-glucuronidase (UniProt accession number alccl 2), Trichodermareesei alpha-glucuronidase (Uniprot accession number Q99024),Talaromyces emersonii alpha-glucuronidase (UniProt accession numberQ8X211), Aspergillus niger alpha-glucuronidase (Uniprot accession numberQ96VVX9), Aspergillus terreus alpha-glucuronidase (SwissProt accessionnumber Q0CJP9), and Aspergillus fumigatus alpha-glucuronidase (SwissProtaccession number Q4VWV45).

The enzymes and proteins used in the methods of the present inventionmay be produced by fermentation of the above-noted microbial strains ona nutrient medium containing suitable carbon and nitrogen sources andinorganic salts, using procedures known in the art (see, e.g., Bennett,J.W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, AcademicPress, Calif., 1991). Suitable media are available from commercialsuppliers or may be prepared according to published compositions (e.g.,in catalogues of the American Type Culture Collection). Temperatureranges and other conditions suitable for growth and enzyme productionare known in the art (see, e.g., Bailey, J. E., and 011is, D. F.,Biochemical Engineering Fundamentals, McGraw-Hill Book Company, NY,1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme or protein. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe enzyme to be expressed or isolated. The resulting enzymes producedby the methods described above may be recovered from the fermentationmedium and purified by conventional procedures.

Fermentation. The fermentable sugars obtained from the hydrolyzedcellulosic material can be fermented by one or more (several) fermentingmicroorganisms capable of fermenting the sugars directly or indirectlyinto a desired fermentation product. “Fermentation” or “fermentationprocess” refers to any fermentation process or any process comprising afermentation step. Fermentation processes also include fermentationprocesses used in the consumable alcohol industry (e.g., beer and wine),dairy industry (e.g., fermented dairy products), leather industry, andtobacco industry. The fermentation conditions depend on the desiredfermentation product and fermenting organism and can easily bedetermined by one skilled in the art.

In the fermentation step, sugars, released from the cellulosic materialas a result of the pretreatment and enzymatic hydrolysis steps, arefermented to a product, e.g., ethanol, by a fermenting organism, such asyeast. Hydrolysis (saccharification) and fermentation can be separate orsimultaneous, as described herein.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be C₆ and/or C₅ fermenting organisms, or a combinationthereof. Both C₆ and C₅ fermenting organisms are well known in the art.Suitable fermenting microorganisms are able to ferment, i.e., convert,sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose,galactose, or oligosaccharides, directly or indirectly into the desiredfermentation product.

Examples of bacterial and fungal fermenting organisms producing ethanolare described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69:627-642.

Examples of fermenting microorganisms that can ferment C6 sugars includebacterial and fungal organisms, such as yeast. Preferred yeast includesstrains of the Saccharomyces spp., preferably Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment C₅ sugars includebacterial and fungal organisms, such as some yeast. Preferred C₅fermenting yeast include strains of Pichia, preferably Pichia stipitis,such as Pichia stipitis CBS 5773; strains of Candida, preferably Candidaboidinii, Candida brassicae, Candida sheatae, Candida diddensii, Candidapseudotropicalis, or Candida utilis.

Other fermenting organisms include strains of Zymomonas, such asZymomonas mobilis; Hansenula, such as Hansenula anomala; Kluyveromyces,such as K. fragilis; Schizosaccharomyces, such as S. pombe; E. coli,especially E. coli strains that have been genetically modified toimprove the yield of ethanol; Clostridium, such as Clostridiumacetobutylicum, Chlostridium thermocellum, and Chlostridiumphytofermentans; Geobacillus sp.; Thermoanaerobacter, such asThermoanaerobacter saccharolyticum; and Bacillus, such as Bacilluscoagulans.

In a preferred aspect, the yeast is a Saccharomyces spp. In a morepreferred aspect, the yeast is Saccharomyces cerevisiae. In another morepreferred aspect, the yeast is Saccharomyces distaticus. In another morepreferred aspect, the yeast is Saccharomyces uvarum. In anotherpreferred aspect, the yeast is a Kluyveromyces. In another morepreferred aspect, the yeast is Kluyveromyces marxianus. In another morepreferred aspect, the yeast is Kluyveromyces fragilis. In anotherpreferred aspect, the yeast is a Candida. In another more preferredaspect, the yeast is Candida boidinii. In another more preferred aspect,the yeast is Candida brassicae. In another more preferred aspect, theyeast is Candida diddensii. In another more preferred aspect, the yeastis Candida pseudotropicalis. In another more preferred aspect, the yeastis Candida utilis. In another preferred aspect, the yeast is aClavispora. In another more preferred aspect, the yeast is Clavisporalusitaniae. In another more preferred aspect, the yeast is Clavisporaopuntiae. In another preferred aspect, the yeast is a Pachysolen. Inanother more preferred aspect, the yeast is Pachysolen tannophilus. Inanother preferred aspect, the yeast is a Pichia. In another morepreferred aspect, the yeast is a Pichia stipitis. In another preferredaspect, the yeast is a Bretannomyces. In another more preferred aspect,the yeast is Bretannomyces clausenii (Philippidis, G. P., 1996,Cellulose bioconversion technology, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, DC, 179-212).

Bacteria that can efficiently ferment hexose and pentose to ethanolinclude, for example, Zymomonas mobilis, Clostridium acetobutylicum,Clostridium thermocellum, Chlostridium phytofermentans, Geobacillus sp.,Thermoanaerobacter saccharolyticum, and Bacillus coagulans (Philippidis,1996, supra).

In a preferred aspect, the bacterium is a Zymomonas. In a more preferredaspect, the bacterium is Zymomonas mobilis. In another preferred aspect,the bacterium is a Clostridium. In another more preferred aspect, thebacterium is Clostridium thermocellum.

Commercially available yeast suitable for ethanol production includes,e.g., ETHANOL RED™ yeast (Fermentis/Lesaffre, USA), FALI™ (Fleischmann'sYeast, USA), SUPERSTART™ and THERMOSACC™ fresh yeast (EthanolTechnology, Wis., USA), BIOFERM™ AFT and XR (NABC-North AmericanBioproducts Corporation, Ga., USA), GERT STRANDTM (Gert Strand AB,Sweden), and FERMIOL™ (DSM Specialties).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (cofermentation) (Chen and Ho, 1993, Cloning andimproving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TALI genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470;WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganismis Saccharomyces cerevisiae. In another preferred aspect, thegenetically modified fermenting microorganism is Zymomonas mobilis. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Escherichia coli. In another preferred aspect, thegenetically modified fermenting microorganism is Klebsiella oxytoca. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Kluyveromyces sp.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedlignocellulose or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, such as about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., inparticular about 32° C. or 50° C., and at about pH 3 to about pH 8, suchas around pH 4-5, 6, or 7.

In a preferred aspect, the yeast and/or another microorganism is appliedto the degraded cellulosic material and the fermentation is performedfor about 12 to about 96 hours, such as typically 24-60 hours. In apreferred aspect, the temperature is preferably between about 20° C. toabout 60° C., more preferably about 25° C. to about 50° C., and mostpreferably about 32° C. to about 50° C., in particular about 32° C. or50° C., and the pH is generally from about pH 3 to about pH 7,preferably around pH 4-7. However, some fermenting organisms, e.g.,bacteria, have higher fermentation temperature optima. Yeast or anothermicroorganism is preferably applied in amounts of approximately 10⁵ to10¹², preferably from approximately 10⁷ to 10¹⁰, especiallyapproximately 2×10⁸ viable cell count per ml of fermentation broth.Further guidance in respect of using yeast for fermentation can be foundin, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P. Lyons and D.R. Kelsall, Nottingham University Press, United Kingdom 1999), which ishereby incorporated by reference.

For ethanol production, following the fermentation the fermented slurryis distilled to extract the ethanol. The ethanol obtained according tothe methods of the invention can be used as, e.g., fuel ethanol,drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation products: A fermentation product can be any substancederived from the fermentation. The fermentation product can be, withoutlimitation, an alcohol (e.g., arabinitol, butanol, ethanol, glycerol,methanol, 1,3-propanediol, sorbitol, and xylitol); an organic acid(e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid, citricacid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaricacid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionicacid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid,oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); aketone (e.g., acetone); an amino acid (e.g., aspartic acid, glutamicacid, glycine, lysine, serine, and threonine); and a gas (e.g., methane,hydrogen (H₂), carbon dioxide (CO2), and carbon monoxide (CO)). Thefermentation product can also be protein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. In a more preferred aspect, thealcohol is arabinitol. In another more preferred aspect, the alcohol isbutanol. In another more preferred aspect, the alcohol is ethanol. Inanother more preferred aspect, the alcohol is glycerol. In another morepreferred aspect, the alcohol is methanol. In another more preferredaspect, the alcohol is 1,3-propanediol. In another more preferredaspect, the alcohol is sorbitol. In another more preferred aspect, thealcohol is xylitol. See, for example, Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira, M.M., and Jonas, R., 2002, The biotechnological production of sorbitol,Appl. Microbiol. Biotechnol. 59: 400-408; Nigam, P., and Singh, D.,1995, Processes for fermentative production of xylitol—a sugarsubstitute, Process Biochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi,N. and Blaschek, H. P., 2003, Production of acetone, butanol and ethanolby Clostridium beijerinckii BA101 and in situ recovery by gas stripping,World Journal of Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. In another more preferred aspect,the ketone is acetone. See, for example, Qureshi and Blaschek, 2003,supra.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example, Richard, A., andMargaritis, A., 2004, Empirical modeling of batch fermentation kineticsfor poly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO2. In another more preferred aspect, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114,1997, Anaerobic digestion of biomass for methane production: A review.

Recovery. The fermentation product(s) can be optionally recovered fromthe fermentation medium using any method known in the art including, butnot limited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol.% can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

Detergent Compositions

The polypeptides having cellulolytic enhancing activity of the presentinvention may be added to and thus become a component of a detergentcomposition.

The detergent composition of the present invention may be formulated,for example, as a hand or machine laundry detergent compositionincluding a laundry additive composition suitable for pre-treatment ofstained fabrics and a rinse added fabric softener composition, or beformulated as a detergent composition for use in general household hardsurface cleaning operations, or be formulated for hand or machinedishwashing operations. Consequently, the present invention also relatesto methods for cleaning or washing a hard surface or laundry, comprisingcontacting the hard surface or the laundry with a detergent compositionof the present invention.

In a specific aspect, the present invention provides a detergentadditive comprising a polypeptide of the invention. The detergentadditive as well as the detergent composition may further comprise oneor more (several) enzymes selected from the group consisting of anamylase, an arabinase, a carbohydrase, a cellulase, a cutinase, agalactanase, a hemicellulase, a laccase, a lipase, a mannanase, anoxidase, a pectinase, a protease, and a xylanase.

one or more enzymes such as a protease, lipase, cutinase, an amylase,carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase,xylanase, oxidase, e.g., a laccase, and/or peroxidase. furthercomprising one or more enzymes selected from the group consisting of acellulase, a protease, a lipase, a cutinase, an amylase, a carbohydrase,a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, andan oxidase

In general the properties of the selected enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Cellulases: Suitable cellulases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Suitable cellulases include cellulases from the genera Bacillus,Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungalcellulases produced from Humicola insolens, Myceliophthora thermophilaand Fusarium oxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263,5,691,178, 5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving color care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. Nos. 5,457,046, 5,686,593,5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Commercially available cellulases include CELLUZYME™, and CAREZYME™(Novozymes A/S), CLAZINASE™, and PURADAX HA™ (Genencor InternationalInc.), and KAC-500(B)™ (Kao Corporation).

Proteases: Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically modified orprotein engineered mutants are included. The protease may be a serineprotease or a metalloprotease, preferably an alkaline microbial proteaseor a trypsin-like protease. Examples of alkaline proteases aresubtilisins, especially those derived from Bacillus, e.g., subtilisinNovo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 andsubtilisin 168 (described in WO 89/06279). Examples of trypsin-likeproteases are trypsin (e.g., of porcine or bovine origin) and theFusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more of the following positions: 27, 36, 57, 76,87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, and274.

Preferred commercially available protease enzymes include ALCALASE198 ,SAVINASE™ PRIMASE™, DURALASE™, ESPERASE™, and KANNASE™ (Novozymes A/S),MAXATASE™, MAXACAL™, MAXAPEM™, PROPERASE™, PURAFECT™, PURAFECT OXP™,FN2™, and FN3™ (Genencor International Inc.).

Lipases: Suitable lipases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful lipases include lipases from Humicola (synonym Thermomyces),e.g., from H. lanuginosa (T. lanuginosus) as described in EP 258 068 andEP 305 216 or from H. insolens as described in WO 96/13580, aPseudomonas lipase, e.g., from P. alcaligenes or P. pseudoalcaligenes(EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g.,from B. subtilis (Dartois et al., 1993, Biochemica et Biophysica Acta,1131: 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO91/16422).

Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

Preferred commercially available lipase enzymes include LIPOLASE™ andLIPOLASE ULTRA™ (Novozymes A/S).

Amylases: Suitable amylases (alpha and/or beta) include those ofbacterial or fungal origin. Chemically modified or protein engineeredmutants are included. Amylases include, for example, alpha-amylasesobtained from Bacillus, e.g., a special strain of Bacilluslicheniformis, described in more detail in GB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597,WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,264, 304, 305, 391, 408, and 444.

Commercially available amylases are DURAMYL™, TERMAMYL™, FUNGAMYL™ andBAN™ (Novozymes A/S), RAPIDASE™ and PURASTAR™ (from GenencorInternational Inc.).

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those ofplant, bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g., from C. cinereus, and variants thereofas those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include GUARDZYME™ (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated, for example, as a granulate, liquid, slurry, etc.Preferred detergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the invention may be in any convenientform, e.g., a bar, a tablet, a powder, a granule, a paste, or a liquid.A liquid detergent may be aqueous, typically containing up to 70% waterand 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid, orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates, or layered silicates (e.g., SKS-6 fromHoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers,and lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of, for example, the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in, for example, WO92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

In the detergent compositions, any enzyme may be added in an amountcorresponding to 0.01-100 mg of enzyme protein per liter of wash liquor,preferably 0.05-5 mg of enzyme protein per liter of wash liquor, inparticular 0.1-1 mg of enzyme protein per liter of wash liquor.

In the detergent compositions, a polypeptide of the present inventionhaving cellulolytic enhancing activity may be added in an amountcorresponding to 0.001-100 mg of protein, preferably 0.005-50 mg ofprotein, more preferably 0.01-25 mg of protein, even more preferably0.05-10 mg of protein, most preferably 0.05-5 mg of protein, and evenmost preferably 0.01-1 mg of protein per liter of wash liquor.

A polypeptide of the invention having cellulolytic enhancing activitymay also be incorporated in the detergent formulations disclosed in WO97/07202, which is hereby incorporated by reference.

Signal Peptide

The present invention also relates to an isolated polynucleotideencoding a signal peptide comprising or consisting of amino acids 1 to17 of SEQ ID NO: 2, amino acids 1 to 19 of SEQ ID NO: 4, amino acids 1to 17 of SEQ ID NO: 6, amino acids 1 to 19 of SEQ ID NO: 8, amino acids1 to 21 of SEQ ID NO: 10, amino acids 1 to 24 of SEQ ID NO: 12, aminoacids 1 to 16 of SEQ ID NO: 14, amino acids 1 to 18 of SEQ ID NO: 16,amino acids 1 to 22 of SEQ ID NO: 18, amino acids 1 to 16 of SEQ ID NO:20, or amino acids 1 to 19 of SEQ ID NO: 22. The polynucleotide mayfurther comprise a gene encoding a protein, which is operably linked tothe signal peptide. The protein is preferably foreign to the signalpeptide.

The present invention also relates to nucleic acid constructs,expression vectors and recombinant host cells comprising such apolynucleotide.

The present invention also relates to methods of producing a protein,comprising: (a) cultivating a recombinant host cell comprising such apolynucleotide; and (b) recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andpolypeptides. The term “protein” also encompasses two or morepolypeptides combined to form the encoded product. The proteins alsoinclude hybrid polypeptides and fused polypeptides.

Preferably, the protein is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.For example, the protein may be an oxidoreductase, transferase,hydrolase, lyase, isomerase, or ligase such as an aminopeptidase,amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase,cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,invertase, laccase, another lipase, mannosidase, mutanase, oxidase,pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolyticenzyme, ribonuclease, transglutaminase or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strains

Thielavia terrestris NRRL 8126 was used as the source of the Family 61polypeptides having cellulolytic enhancing activity. Aspergillus oryzaeJaL355 strain (WO 2002/40694) was used for expression of the Thielaviaterrestris Family 61 genes encoding the polypeptides having cellulolyticenhancing activity.

Media and Solutions

PDA plates were composed of 39 g of potato dextrose agar and distilledwater to 1 liter.

NNCYP medium was composed of 5.0 g of NaNO₃, 3.0 g of NH₄Cl, 2.0 g ofMES, 2.5 g of citric acid, 0.2 g of CaCl₂ 2H₂O, 1.0 g of Bacto Peptone,5.0 g of yeast extract, 0.2 g of MgSO₄ 7H₂O, 4.0 g of K2HPO₄, 1.0 ml ofCOVE trace elements solution, 2.5 g of glucose, and distilled water to 1liter.

Minimal medium (MM) plates were composed of 6 g of NaNO₃, 0.52 g of KCl,1.52 g of KH₂PO₄, 1 ml of COVE trace elements solution, 20 g of Nobleagar, 20 ml of 50% glucose, 2.5 ml of MgSO₄·7H₂O, 20 ml of a 0.02%biotin solution, and distilled water to 1 liter.

COVE trace elements solution was composed of 0.04 g of Na₂B₄O₇·10H₂O,0.4 g of CuSO₄·5H₂O, 1.2 g of FeSO₄·7H₂O, 0.7 g of MnSO₄·H₂O, 0.8 g ofNa₂MoO₂·2H₂O, 10 g of ZnSO₄·7H₂O, and distilled water to 1 liter.

M410 medium was composed of 50 g of maltose, 50 g of glucose, 2 g ofMgSO₄·7H₂O, 4 g of citric acid anhydrous powder, 2 g of KH₂PO₄, 8 g ofyeast extract, 2 g of urea, 0.5 g of CaCl₂, 0.5 ml of AMG trace metalssolution, and distilled water to 1 liter.

AMG trace metals solution was composed of 14.3 g of ZnSO₄·7H₂O, 2.5 g ofCuSO₄·5H₂O, 0.5 g of NiCl₂·6H₂O, 13.8 g of FeSO₄·7H₂O, 8.5 g ofMnSO₄·7H₂O, 3 g of citric acid, and distilled water to 1 liter.

Example 1 Source of DNA Sequence Information for Thielavia terrestrisNRRL 8126

Genomic sequence information was generated by the U.S. Department ofEnergy Joint Genome Institute (JGI). A preliminary assembly of thegenome was downloaded from JGI and analyzed using the Pedant-Pro™Sequence Analysis Suite (Biomax Informatics AG, Martinsried, Germany).Gene models constructed by the software were used as a starting pointfor detecting GH61 homologues in the genome. More precise gene modelswere constructed manually using multiple known GH61 protein sequences asa guide.

Example 2 Thielavia terrestris NRRL 8126 Genomic DNA Extraction

To generate genomic DNA for PCR amplification, Thielavia terrestris NRRL8126 was grown in 50 ml of NNCYP medium supplemented with 1% glucose ina baffled shake flask at 42° C. and 200 rpm for 24 hours. Mycelia wereharvested by filtration, washed twice in TE (10 mM Tris-1 mM EDTA), andfrozen under liquid nitrogen. A pea-size piece of frozen mycelia wassuspended in 0.7 ml of 1% lithium dodecyl sulfate in TE and disrupted byagitation with an equal volume of 0.1 mm zirconia/silica beads (BiospecProducts, Inc., Bartlesville, Okla., USA) for 45 seconds in a FastPrepFP120 (ThermoSavant, Holbrook, N.Y., USA). Debris was removed bycentrifugation at 13,000× g for 10 minutes and the cleared supernatantwas brought to 2.5 M ammonium acetate and incubated on ice for 20minutes. After the incubation period, the nucleic acids wereprecipitated by addition of 2 volumes of ethanol. After centrifugationfor 15 minutes in a microfuge at 4° C., the pellet was washed in 70%ethanol and air dried. The DNA was resuspended in 120 μl of 0.1× TE andincubated with 1 μl of DNase-free RNase A at 37° C. for 20 minutes.Ammonium acetate was added to 2.5 M and the DNA was precipitated with 2volumes of ethanol. The pellet was washed in 70% ethanol, air dried, andresuspended in TE buffer.

Example 3 Construction of an Aspergillus oryzae Expression VectorContaining Thielavia terrestris NRRL 8126 Genomic Sequence Encoding aFamily GH61J polypeptide having Cellulolytic Enhancing Activity

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Thielavia terrestris NRRL 8126 gh61j gene from the genomicDNA prepared in Example 2. An IN-FUSION™ Cloning Kit (BD Biosciences,Palo Alto, Calif., USA) was used to clone the fragment directly into theexpression vector pAlLo2 (WO 2004/099228), without the need forrestriction digests and ligation.

Ttgh1j-F (065367): (SEQ ID NO: 23)5′-ACTGGATTTACCATGAAGTTCTCACTGGTGTC-3′ Ttgh61j-R (065368):(SEQ ID NO: 24) 5′-TCACCTCTAGTTAATTAATCAGCAGGAGATCGGGGCGG-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAlLo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncomposed of 100 ng of Thielavia terrestris NRRL 8126 genomic DNA, PfxAmplification Buffer (Invitrogen, Carlsbad, Calif., USA), 0.4 mM each ofdATP, dTTP, dGTP, and dCTP, 1 mM MgCl₂, and 2.5 units of Pfx DNApolymerase (Invitrogen, Carlsbad, Calif., USA) in a final volume of 50μl. The amplification was performed using an EPPENDORF® MASTERCYCLER®5333 (Eppendorf Scientific, Inc., Westbury, N.Y., USA) programmed for 1cycle at 98° C. for 3 minutes; and 30 cycles each at 98° C. for 30seconds, 60° C. for 30 seconds, and 72° C. for 1.5 minutes. The heatblock then went to a 4° C. soak cycle.

The reaction products were isolated on a 1.0% agarose gel using 40 mMTris base-20 mM sodium acetate-1 mM disodium EDTA (TAE) buffer where a908 bp product band was excised from the gel and purified using aMINELUTE® Gel Extraction Kit (QIAGEN Inc., Valencia, Calif., USA)according to the manufacturer's instructions. The fragment was thencloned into Nco I and Pac I digested pAlLo2 using an IN-FUSION™ CloningKit resulting in pSMai207 in which transcription of the Thielaviaterrestris gh61j gene was under the control of a NA2-tpi promoter (amodified promoter from the gene encoding neutral alpha-amylase inAspergillus niger in which the untranslated leader has been replaced byan untranslated leader from the gene encoding triose phosphate isomerasein Aspergillus nidulans). The ligation reaction (50 μl) was composed of1× IN-FUSION™ Buffer (BD Biosciences, Palo Alto, Calif., USA), 1× BSA(BD Biosciences, Palo Alto, Calif., USA), 1 μl of IN-FUSION™ enzyme(diluted 1:10) (BD Biosciences, Palo Alto, Calif., USA), 100 ng ofpAlLo2 digested with Nco I and Pac I, and 50 ng of the Thielaviaterrestris gh61j purified PCR product. The reaction was incubated atroom temperature for 30 minutes. One μl of the reaction was used totransform E. coli XL10 SOLOPACK® Gold Supercompetent cells (Stratagene,La Jolla, Calif., USA). An E. coli transformant containing pSMai207 wasdetected by restriction digestion and plasmid DNA was prepared using aBIOROBOT® 9600 (QIAGEN Inc., Valencia, Calif., USA). The Thielaviaterrestris gh61j insert in pSMai207 was confirmed by DNA sequencing.

The same 908 bp Thielavia terrestris gh61j PCR fragment was also clonedinto pCR02.1-TOPO vector (Invitrogen, Carlsbad, Calif., USA) using aTOPO® TA CLONING® Kit (Invitrogen, Carlsbad, Calif., USA), to generatepSMai216. The Thielavia terrestris gh61j insert was confirmed by DNAsequencing. E. coli pSMai216 was deposited with the AgriculturalResearch Service Patent Culture Collection, Northern Regional ResearchCenter, Peoria, Ill., USA, on Aug. 3, 2009 and assigned accession numberNRRL B-50301.

Example 4 Characterization of the Thielavia terrestris NRRL 8126 GenomicSequence Encoding a GH61J Polypeptide having Cellulolytic-enhancingActivity

DNA sequencing of the Thielavia terrestris NRRL 8126 gh61j genomic clonewas performed with an Applied Biosystems Model 3700 Automated DNASequencer using version 3.1 BIG-DYE™ terminator chemistry (AppliedBiosystems, Inc., Foster City, Calif., USA) and dGTP chemistry (AppliedBiosystems, Inc., Foster City, Calif., USA) and primer walking strategy.Nucleotide sequence data were scrutinized for quality and all sequenceswere compared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash., USA). The sequence obtainedwas identical to the sequence from JGI.

The nucleotide sequence (SEQ ID NO: 1) and deduced amino acid sequence(SEQ ID NO: 2) of the Thielavia terrestris gh61j gene are shown inFIG. 1. The coding sequence is 878 bp including the stop codon and isinterrupted by introns of 66 and 71 bp. The encoded predicted protein is246 amino acids. The % G+C of the coding sequence of the gene (includingintrons) is 63% G+C and the mature polypeptide coding sequence is 63%.Using the SignalP program (Nielsen et al., 1997, Protein Engineering 10:1-6), a signal peptide of 17 residues was predicted. The predictedmature protein contains 229 amino acids with a predicted molecular massof 24.5 kDa and an isoelectric pH of 7.85.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program ofEMBOSS with gap open penalty of 10, gap extension penalty of 0.5, andthe EBLOSUM62 matrix. The alignment showed that the deduced amino acidsequence of the Thielavia terrestris gene encoding the GH61J polypeptidehaving cellulolytic-enhancing activity shares 57.7% identity (excludinggaps) to the deduced amino acid sequence of a predicted GH61 familyprotein from Humicola insolens (accession numbers geneseqp:ADM97935).

Example 5 Expression of Thielavia terrestris NRRL 8126 Family 61Glycosyl hydrolase 61j Gene in Aspergillus oryzae JaL355

Aspergillus oryzae JaL355 (WO 2002/40694) protoplasts were preparedaccording to the method of Christensen et al., 1988, Bio/Technology 6:1419-1422, which were transformed with approximately 2 μg of pSMai207.The transformation yielded about 30 transformants. Ten transformantswere isolated to individual Minimal Medium plates.

Confluent Minimal Medium plates of each of the transformants were washedwith 5 ml of 0.01% TWEEN® 20 and inoculated separately into 25 ml ofM410 medium in 125 ml glass shake flasks and incubated at 34° C., 250rpm. After 5 days incubation, 5 μl of supernatant from each culture wereanalyzed on CRITERION® Tris-HCl gels (Bio-Rad Laboratories, Hercules,Calif., USA) with a CRITERION® Cell (Bio-Rad Laboratories, Hercules,Calif., USA), according to the manufacturer's instructions. Theresulting gels were stained with BIO-SAFE™ Coomassie Stain (Bio-RadLaboratories, Hercules, Calif., USA). SDS-PAGE profiles of the culturesshowed that the majority of the transformants had an expected 24 KDaband size. A confluent plate of transformant 3 was washed with 10 ml of0.01% TWEEN® 80 and inoculated into a 2 liter Fernbach containing 500 mlof M410 medium to generate broth for characterization of the enzyme. Theculture was harvested on day 5 and filtered using a 0.22 μm EXPRESS™PLUS Membrane (Millipore, Billerica, Mass., USA).

Example 6 Construction of an Aspergillus oryzae Expression VectorContaining Thielavia terrestris NRRL 8126 Genomic Sequence Encoding aFamily GH61K Polypeptide having Cellulolytic Enhancing Activity

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Thielavia terrestris NRRL 8126 gh61 k gene from the genomicDNA prepared in Example 2. An IN-FUSION™ Cloning Kit was used to clonethe fragment directly into the expression vector pAlLo2, without theneed for restriction digests and ligation.

Ttgh1k-F (065465): (SEQ ID NO: 25)5′-ACTGGATTTACCATGAGGACGACATTCGCCGCCGCGT-3′ Ttgh61k-R (065466):(SEQ ID NO: 26) 5′-TCACCTCTAGTTAATTAACTAAGAAGAAGGGGCGCACT-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAlLo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncomposed of 100 ng of Thielavia terrestris NRRL 8126 genomic DNA, PfxAmplification Buffer, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, 1 mMMgCl₂, and 2.5 units of Pfx DNA polymerase in a final volume of 50 μl.The amplification was performed using an EPPENDORF® MASTERCYCLER® 5333programmed for 1 cycle at 98° C. for 3 minutes; and 30 cycles each at98° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 1.5minutes. The heat block then went to a 4° C. soak cycle.

The reaction products were isolated on a 1.0% agarose gel using TAEbuffer where a 1283 bp product band was excised from the gel andpurified using a MINELUTEO Gel Extraction Kit according to themanufacturer's instructions. The fragment was then cloned into Nco I andPac I digested pAlLo2 using an 1N-FUSION™ Cloning Kit resulting inpSMai208 in which transcription of the Thielavia terrestris gh61 k genewas under the control of a NA2-tpi promoter. The ligation reaction (50μl) was composed of 1× IN-FUSION™ Buffer, 1× BSA, 1 μl of IN-FUSION™enzyme (diluted 1:10), 100 ng of pAlLo2 digested with Nco I and Pac I,and 50 ng of the Thielavia terrestris gh6lk purified PCR product. Thereaction was incubated at room temperature for 30 minutes. One μl of thereaction was used to transform E. coli XL10 SOLOPACK® GoldSupercompetent cells. An E. coli transformant containing pSMai208 wasdetected by restriction digestion and plasmid DNA was prepared using aBIOROBOT® 9600. The Thielavia terrestris gh61 k insert in pSMai208 wasconfirmed by DNA sequencing.

The same 1283 bp Thielavia terrestris gh61 k PCR fragment was alsocloned into pCR®2.1-TOPO vector using a TOPO TA CLONING® Kit, togenerate pSMai217. The Thielavia terrestris gh61 k insert was confirmedby DNA sequencing. E. coli pSMai217 was deposited with the AgriculturalResearch Service Patent Culture Collection, Northern Regional ResearchCenter, Peoria, Ill., USA, on Aug. 3, 2009 and assigned accession numberNRRL B-50302.

Example 7 Characterization of the Thielavia terrestris NRRL 8126 GenomicSequence Encoding a GH61K Polypeptide having Cellulolytic-enhancingActivity

DNA sequencing of the Thielavia terrestris NRRL 8126 gh61 k genomicclone was performed with an Applied Biosystems Model 3700 Automated DNASequencer using version 3.1 BIG-DYE™ terminator chemistry and dGTPchemistry and primer walking strategy. Nucleotide sequence data werescrutinized for quality and all sequences were compared to each otherwith assistance of PHRED/PHRAP software. The sequence obtained wasidentical to the sequence from the JGI.

The nucleotide sequence (SEQ ID NO: 3) and deduced amino acid sequence(SEQ ID NO: 4) of the Thielavia terrestris gh61 k gene are shown in FIG.2. The coding sequence is 1253 bp including the stop codon and isinterrupted by introns of 96, 84 and 68 bp. The encoded predictedprotein is 334 amino acids. The % G+C of the coding sequence of the gene(including introns) is 66.6% G+C and the mature polypeptide codingsequence is 69.3%. Using the SignalP program (Nielsen et al., 1997,supra), a signal peptide of 19 residues was predicted. The predictedmature protein contains 315 amino acids with a predicted molecular massof 31.7 kDa and an isoelectric pH of 6.68.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, supra) as implemented in the Needle program of EMBOSS with gapopen penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62matrix. The alignment showed that the deduced amino acid sequence of theThielavia terrestris gene encoding the GH61K polypeptide havingcellulolytic-enhancing activity shares 64.8% identity (excluding gaps)to the deduced amino acid sequence of a predicted beta-glucosidaseprotein from Penicillium brasilianum (accession numbers geneseqpAWW27060).

Example 8 Expression of Thielavia terrestris NRRL 8126 Family 61Glycosyl hydrolase 61k Gene in Aspergillus oryzae JaL355

Aspergillus oryzae JaL355 (WO 2002/40694) protoplasts were preparedaccording to the method of Christensen et al., 1988, supra, which weretransformed with approximately 2 μg of pSMai208. The transformationyielded about 25 transformants. Ten transformants were isolated toindividual Minimal Medium plates.

Confluent Minimal Medium plates of each of the transformants were washedwith 5 ml of 0.01% TWEEN® 20 and inoculated separately into 25 ml ofM410 medium in 125 ml glass shake flasks and incubated at 34° C., 250rpm. After 5 days incubation, 5 μl of supernatant from each culture wereanalyzed on CRITERION® Tris-HCl gels with a CRITERION® Cell, accordingto the manufacturer's instructions. The resulting gels were stained withBIO-SAFE™ Coomassie Stain. SDS-PAGE profiles of the cultures showed thatthe majority of the transformants had an expected 32 KDa band size. Aconfluent plate of transformant 5 was washed with 10 ml of 0.01% TWEEN®80 and inoculated into a 2 liter Fernbach containing 500 ml of M410medium to generate broth for characterization of the enzyme. The culturewas harvested on day 5 and filtered using a 0.22 μm EXPRESS™ PLUSMembrane.

Example 9 Construction of an Aspergillus oryzae Expression VectorContaining Thielavia terrestris NRRL 8126 Genomic Sequence Encoding aFamily GH61L polypeptide having Cellulolytic Enhancing Activity

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Thielavia terrestris NRRL 8126 gh6l/gene from the genomicDNA prepared in Example 2. An IN-FUSION™ Cloning Kit was used to clonethe fragment directly into the expression vector pAlLo2, without theneed for restriction digests and ligation.

Ttgh1I-F1 (066276): (SEQ ID NO: 27)5′-ACTGGATTTACCATGAAGCTGAGCGTTGCCATCGCC-3′ Ttgh61I-R (065736):(SEQ ID NO: 28) 5′-TCACCTCTAGTTAATTAATTAGCACGTCTCAGCCGGCG-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAlLo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncomposed of 100 ng of Thielavia terrestris NRRL 8126 genomic DNA, PfxAmplification Buffer, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, 1 mMMgCl₂, and 2.5 units of Pfx DNA polymerase in a final volume of 50 μl.The amplification was performed using an EPPENDORF® MASTERCYCLER® 5333programmed for 1 cycle at 98° C. for 3 minutes; and 30 cycles each at98° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 1.5minutes. The heat block then went to a 4° C. soak cycle.

The reaction products were isolated on a 1.0% agarose gel using TAEbuffer where a 828 bp product band was excised from the gel and purifiedusing a MINELUTE® Gel Extraction Kit according to the manufacturer'sinstructions. The fragment was then cloned into Nco I and Pac I digestedpAlLo2 using an IN-FUSION™ Cloning Kit resulting in pSMai209 in whichtranscription of the Thielavia terrestris gh61l gene was under thecontrol of a NA2-tpi promoter. The ligation reaction (50 μl) wascomposed of 1× IN-FUSION™ Buffer, 1× BSA, 1 μl of IN-FUSION™ enzyme(diluted 1:10), 100 ng of pAlLo2 digested with Nco I and Pac I, and 50ng of the Thielavia terrestris gh61l purified PCR product. The reactionwas incubated at room temperature for 30 minutes. One μl of the reactionwas used to transform E. coli XL10 SOLOPACK® Gold Supercompetent cells.An E. coli transformant containing pSMai212 was detected by restrictiondigestion and plasmid DNA was prepared using a BIOROBOT® 9600. TheThielavia terrestris gh61l insert in pSMai212 was confirmed by DNAsequencing.

The same 828 bp Thielavia terrestris gh61l PCR fragment was also clonedinto pCR®2.1-TOPO vector using a TOPO TA CLONING® Kit, to generatepSMai218. The Thielavia terrestris gh61l insert was confirmed by DNAsequencing. E. coli pSMai218 was deposited with the AgriculturalResearch Service Patent Culture Collection, Northern Regional ResearchCenter, Peoria, Ill., USA, on Aug. 3, 2009 and assigned accession numberNRRL B-50303.

Example 10 Characterization of the Thielavia terrestris NRRL 8126Genomic Sequence Encoding a GH61L Polypeptide havingCellulolytic-enhancing Activity

DNA sequencing of the Thielavia terrestris NRRL 8126 gh61l genomic clonewas performed with an Applied Biosystems Model 3700 Automated DNASequencer using version 3.1 BIG-DYE™ terminator chemistry and dGTPchemistry and primer walking strategy. Nucleotide sequence data werescrutinized for quality and all sequences were compared to each otherwith assistance of PHRED/PHRAP software. The sequence obtained wasidentical to the sequence from the JGI.

The nucleotide sequence (SEQ ID NO: 5) and deduced amino acid sequence(SEQ ID NO: 6) of the Thielavia terrestris gh61l gene are shown in FIG.3. The coding sequence is 798 bp including the stop codon and isinterrupted by introns of 55 and 59 bp. The encoded predicted protein is227 amino acids. The % G+C of the coding sequence of the gene (includingintrons) is 60.8% G+C and the mature polypeptide coding sequence is62.6%. Using the SignalP program (Nielsen et al., 1997, supra), a signalpeptide of 17 residues was predicted. The predicted mature proteincontains 210 amino acids with a predicted molecular mass of 22.6 kDa andan isoelectric pH of 8.84.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, supra) as implemented in the Needle program of EMBOSS with gapopen penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62matrix. The alignment showed that the deduced amino acid sequence of theThielavia terrestris gene encoding the GH61L polypeptide havingcellulolytic-enhancing activity shares 59.2% identity (excluding gaps)to the deduced amino acid sequence of a predicted GH61 family proteinfrom Thielavia terrestris (accession numbers geneseqp ADM97933).

Example 11 Expression of Thielavia terrestris NRRL 8126 Family 61Glycosyl Hydrolase 61/ gene in Aspergillus oryzae JaL355

Aspergillus oryzae JaL355 (WO 2002/40694) protoplasts were preparedaccording to the method of Christensen et al., 1988, supra, which weretransformed with approximately 2 μg of pSMai212. The transformationyielded about 17 transformants. Seventeen transformants were isolated toindividual Minimal Medium plates.

Confluent Minimal Medium plates of each of the transformants were washedwith 5 ml of 0.01% TWEEN® 20 and inoculated separately into 25 ml ofM410 medium in 125 ml glass shake flasks and incubated at 34° C., 250rpm. After 5 days incubation, 5 μl of supernatant from each culture wereanalyzed on CRITERION® Tris-HCl gels with a CRITERION® Cell, accordingto the manufacturer's instructions. The resulting gels were stained withBIO-SAFE™ Coomassie Stain. SDS-PAGE profiles of the cultures showed thatthe majority of the transformants had an expected 23 KDa band size. Aconfluent plate of transformant 14 was washed with 10 ml of 0.01% TWEEN®80 and inoculated into a 2 liter Fernbach containing 500 ml of M410medium to generate broth for characterization of the enzyme. The culturewas harvested on day 5 and filtered using a 0.22 μm EXPRESS™ PLUSMembrane.

Example 12 Hydrolysis of Pretreated Corn Stover is Enhanced by Thielaviaterrestris NRRL 8126 GH61J, GH61K, and GFH61L Polypeptides havingCellulolytic Enhancing Activity

Culture broth was prepared as described in Examples 5, 8, and 11 andconcentrated approximately 20-fold using an Amicon ultrafiltrationdevice (Millipore, Bedford, Mass., USA, 10 kDa polyethersulfonemembrane, 40 psi, 4° C.). Protein concentration was estimated bydensitometry following SDS-PAGE and Coomassie blue staining. Corn stoverwas pretreated and prepared as an assay substrate as described in WO2005/074647 to generate pretreated corn stover (PCS). The base cellulasemixture used to assay enhancing activity was prepared from Trichodermareesei strain SMA135 (WO 2008/057637).

Hydrolysis of PCS was conducted using 1.6 ml deep-well plates (Axygen,Santa Clara, Calif., USA) using a total reaction volume of 1.0 ml and aPCS concentration of 50 mg/ml in 1 mM manganese sulfate-50 mM sodiumacetate, pH 5.0. The T. terrestris polypeptides (GH61J, GH61K, andGFH61L) were separately added to the base cellulase mixture atconcentrations ranging from 0 to 25% or 0 to 32% of the proteinconcentration of the base cellulase mixture. Incubation was at 50° C.for 72 hours. Assays were performed in triplicate. Aliquots werecentrifuged, and the supernatant liquid was filtered by centrifugation(MULTISCREEN® HV 0.45 μm, Millipore, Billerica, Mass., USA) at 3000 rpmfor 10 minutes using a plate centrifuge (SORVALL® RT7, Thermo FisherScientific, Waltham, Mass., USA). When not used immediately, filteredhydrolysate aliquots were frozen at −20° C. Sugar concentrations ofsamples diluted in 0.005 M H₂SO₄ with 0.05% w/w benzoic acid weremeasured after elution by 0.005 M H₂SO₄ with 0.05% w/w benzoic acid at aflow rate of 0.6 ml/minute from a 4.6×250 mm AMINEX® HPX-87H column(Bio-Rad Laboratories, Inc., Hercules, Calif., USA) at 65° C. withquantitation by integration of glucose and cellobiose signals fromrefractive index detection (CHEMSTATION®, AGILENT® 1100 HPLC, AgilentTechnologies, Santa Clara, Calif., USA) calibrated by pure sugar samples(Absolute Standards Inc., Hamden, Conn., USA). The resultant equivalentswere used to calculate the percentage of cellulose conversion for eachreaction. The degree of cellulose conversion to glucose plus cellobiosesugars (conversion, %) was calculated using the following equation:

Conversion (%)=(glucose+cellobiose×1.053) (mg/ml)×100×162/(Cellulose(mg/ml)×180)=(glucose+cellobiose×1.053) (mg/ml)×100/(Cellulose(mg/ml)×1.111)

In this equation the factor 1.111 reflects the weight gain in convertingcellulose to glucose, and the factor 1.053 reflects the weight gain inconverting cellobiose to glucose. Cellulose in PCS was determined by alimit digest of PCS to release glucose and cellobiose.

The results of adding increasing amounts of Thielavia terrestrispolypeptides separately to the base cellulase mix are shown in FIG. 4.Addition of each of the T. terrestris GH61J and GH61K polypeptidesprovided a stimulation factor of 1.14 and 1.13, respectively, at a 25%addition level. T. terrestris GH61L polypeptide provided a stimulationfactor of 1.13 at a 32% addition level.

Example 13 Construction of an Aspergillus oryzae expression vectorcontaining Thielavia terrestris NRRL 8126 Genomic Sequence Encoding aFamily GH61M Polypeptide having Cellulolytic Enhancing Activity

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Thielavia terrestris NRRL 8126 gh6lm gene from the genomicDNA prepared in Example 2. An IN-FUSION™ Cloning Kit was used to clonethe fragment directly into the expression vector pAlLo2 (WO2004/099228), without the need for restriction digests and ligation.

Ttgh1m-F1 (063567): (SEQ ID NO: 29)5′-ACTGGATTTACCATGAAGCTGTCATCCCAGCTCGCC-3′ Ttgh61m-R1 (063568):(SEQ ID NO: 30) 5′-TCACCTCTAGTTAATTAACTAGCACTGAAAGACCGCCG-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAlLo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncomposed of 100 ng of Thielavia terrestris NRRL 8126 genomic DNA, PfxAmplification Buffer, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, 1 mMMgCl₂, and 2.5 units of Pfx DNA polymerase in a final volume of 50 μl.The amplification was performed using an EPPENDORF® MASTERCYCLER® 5333programmed for 1 cycle at 98° C. for 3 minutes; and 30 cycles each at98° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 1.5minutes. The heat block then went to a 4° C. soak cycle.

The reaction products were isolated on a 1.0% agarose gel using TAEbuffer where a 1007 bp product band was excised from the gel andpurified using a MINELUTE® Gel Extraction Kit according to themanufacturer's instructions. The fragment was then cloned into Nco I andPac I digested pAlLo2 using an IN-FUSION™ Cloning Kit resulting inpSMai197 (FIG. 5) in which transcription of the Thielavia terrestrisgh61m gene was under the control of the NA2-tpi promoter. The ligationreaction (50 μl) was composed of 1× IN-FUSION™ Buffer, 1× BSA, 1 μl ofIN-FUSION™ enzyme (diluted 1:10), 100 ng of pAlLo2 digested with Nco Iand Pac I, and 50 ng of the Thielavia terrestris gh61m purified PCRproduct. The reaction was incubated at room temperature for 30 minutes.One μl of the reaction was used to transform E. coli XL10 SOLOPACK® GoldSupercompetent cells. An E. coli transformant containing pSMai197 wasdetected by restriction digestion and plasmid DNA was prepared using aBIOROBOT® 9600. The Thielavia terrestris gh61m insert in pSMai197 wasconfirmed by DNA sequencing.

The same 1007 bp Thielavia terrestris gh61m PCR fragment was also clonedinto pCR®2.1-TOPO vector using a TOPO TA CLONING® Kit to generatepSMai213. The Thielavia terrestris gh61m insert was confirmed by DNAsequencing. E. coli pSMai213 was deposited with the AgriculturalResearch Service Patent Culture Collection, Northern Regional ResearchCenter, Peoria, Ill., USA, on Aug. 3, 2009 and assigned accession numberNRRL B-50300.

Example 14 Characterization of the Thielavia terrestris NRRL 8126Genomic Sequence Encoding a GH61M Polypeptide havingCellulolytic-enhancing Activity

DNA sequencing of the Thielavia terrestris NRRL 8126 gh61m genomic clonewas performed with an Applied Biosystems Model 3700 Automated DNASequencer using version 3.1 BIG-DYE™ terminator chemistry and dGTPchemistry and primer walking strategy. Nucleotide sequence data werescrutinized for quality and all sequences were compared to each otherwith assistance of PHRED/PHRAP software. The sequence obtained wasidentical to the sequence from the JGI.

The nucleotide sequence (SEQ ID NO: 7) and deduced amino acid sequence(SEQ ID NO: 8) of the Thielavia terrestris gh61m gene are shown in FIG.6. The coding sequence is 977 bp including the stop codon and isinterrupted by introns of 85, 96 and 124 bp. The encoded predictedprotein is 223 amino acids. The % G+C of the coding sequence of the gene(including introns) is 62.6% G+C and the mature polypeptide codingsequence is 62.2%. Using the SignalP program (Nielsen et al., 1997,supra), a signal peptide of 19 residues was predicted. The predictedmature protein contains 204 amino acids with a predicted molecular massof 22.2 kDa and an isoelectric pH of 6.58.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, supra) as implemented in the Needle program of EMBOSS with gapopen penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62matrix. The alignment showed that the deduced amino acid sequence of theThielavia terrestris gene encoding the GH61M polypeptide havingcellulolytic-enhancing activity shares 76.5% identity (excluding gaps)to the deduced amino acid sequence of a predicted GH61 family proteinfrom Podospora anserina (accession numbers UniProt B2ADY5).

Example 15 Expression of Thielavia terrestris NRRL 8126 Family 61Glycosyl Hydrolase 61m Gene in Aspergillus oryzae JaL355

Aspergillus oryzae JaL355 (WO 2002/40694) protoplasts were preparedaccording to the method of Christensen et al., 1988, supra, which weretransformed with approximately 2 μg of pSMai197. The transformationyielded about 17 transformants. Ten transformants were isolated toindividual Minimal Medium plates.

Confluent Minimal Medium plates of each of the transformants were washedwith 5 ml of 0.01% TWEEN® 20 and inoculated separately into 25 ml ofM410 medium in 125 ml glass shake flasks and incubated at 34° C., 250rpm. After 5 days incubation, 5 μl of supernatant from each culture wereanalyzed on CRITERION® Tris-HCl gels with a CRITERION® Cell, accordingto the manufacturer's instructions. The resulting gels were stained withBIO-SAFE™ Coomassie Stain. SDS-PAGE profiles of the cultures showed thatthe majority of the transformants had an expected 22 kDa band size. Aconfluent plate of transformant 9 was washed with 10 ml of 0.01% TWEEN®80 and inoculated into a 2 liter Fernbach containing 500 ml of M410medium to generate broth for characterization of the enzyme. The culturewas harvested on day 5 and filtered using a 0.22 μm EXPRESS™ PLUSMembrane.

Example 16 Hydrolysis of Pretreated Corn Stover is Enhanced by Thielaviaterrestris NRRL 8126 GH61M Polypeptide having Cellulolytic EnhancingActivity

Culture broth was prepared as described in Example 15 and concentratedapproximately 20-fold using an Amicon ultrafiltration device (Millipore,Bedford, Mass., USA, 10 kDa polyethersulfone membrane, 40 psi, 4° C.).Protein concentration was estimated by densitometry following SDS-PAGEand Coomassie blue staining. Corn stover was pretreated and prepared asan assay substrate as described in WO 2005/074647 to generate pretreatedcorn stover (PCS). The base cellulase mixture used to assay enhancingactivity was prepared from Trichoderma reesei strain SMA135 (WO2008/057637).

Hydrolysis of PCS was conducted using 1.6 ml deep-well plates (Axygen,Santa Clara, Calif., USA) using a total reaction volume of 1.0 ml and aPCS concentration of 50 mg/ml in 1 mM manganese sulfate-50 mM sodiumacetate, pH 5.0. The T. terrestris polypeptide (GH61M) was added to thebase cellulase mixture at concentrations ranging from 0 to 25% of theprotein concentration of the base cellulase mixture. Incubation was at50° C. for 72 hours. Assays were performed in triplicate. Aliquots werecentrifuged, and the supernatant liquid was filtered by centrifugation(MULTISCREEN® HV 0.45 μm) at 3000 rpm for 10 minutes using a platecentrifuge (SORVALL® RT7, Thermo Fisher Scientific, Waltham, Mass.,USA). When not used immediately, filtered hydrolysate aliquots werefrozen at −20° C. Sugar concentrations of samples diluted in 0.005 MH₂SO₄ with 0.05% w/w benzoic acid were measured after elution by 0.005 MH₂SO₄ with 0.05% w/w benzoic acid at a flow rate of 0.6 ml/minute from a4.6×250 mm AMINEX® HPX-87H column at 65° C. with quantitation byintegration of glucose and cellobiose signals from refractive indexdetection calibrated by pure sugar samples (Absolute Standards Inc.,Hamden, Conn., USA). The resultant equivalents were used to calculatethe percentage of cellulose conversion for each reaction. The degree ofcellulose conversion to glucose plus cellobiose sugars (conversion, %)was calculated using the following equation:

Conversion (%)=(glucose+cellobiose×1.053) (mg/ml)×100×162/(Cellulose(mg/ml)×180)=(glucose+cellobiose×1.053) (mg/ml)×100/(Cellulose(mg/ml)×1.111)

In this equation the factor 1.111 reflects the weight gain in convertingcellulose to glucose, and the factor 1.053 reflects the weight gain inconverting cellobiose to glucose. Cellulose in PCS was determined by alimit digest of PCS to release glucose and cellobiose.

The results of adding increasing amounts of the T. terrestris GH61Mpolypeptide to the base cellulase mix are shown in FIG. 7. Addition ofthe T. terrestris GH61M polypeptide provided a stimulation factor of1.27 at a 25% addition level.

Example 17 Construction of an Aspergillus oryzae Expression Vector forthe Thielavia terrestris Family GH61N Gene

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Thielavia terrestris Family GH61N gene from the genomic DNAprepared in Example 2. An IN-FUSION™ Cloning Kit was used to clone thefragment directly into the expression vector, pAlLo2, without the needfor restriction digests and ligation.

Forward primer: (SEQ ID NO: 31) 5′-ACTGGATTTACCATGCCTTCTTTCGCCTCCAA-3′Reverse primer: (SEQ ID NO: 32)5′-TCACCTCTAGTTAATTAATCAGTTTGCCTCCTCAGCCC-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAlLo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 1 μg of Thielavia terrestris genomic DNA, 1× ADVANTAGE®GC-Melt LA Buffer (BD Biosciences, Palo Alto, Calif., USA), 1μl of 10 mMblend of dATP, dTTP, dGTP, and dCTP, and 1.25 units of ADVANTAGE® GCGenomic LA Polymerase Mix (BD Biosciences, Palo Alto, Calif., USA), in afinal volume of 25 μl. The amplification conditions were one cycle at94° C. for 1 minute; and 30 cycles each at 94° C. for 30 seconds, 60.5°C. for 30 seconds, and 72° C. for 1 minute. The heat block was then heldat 72° C. for 5 minutes followed by a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TAE buffer where an approximately 1.1 kb product band was excisedfrom the gel and purified using a MINELUTE® Gel Extraction Kit accordingto the manufacturer's instructions.

The fragment was then cloned into pAlLo2 using an IN-FUSION™ CloningKit. The vector was digested with Nco I and Pac I. The fragment waspurified by 1.0% agarose gel electrophoresis using TAE buffer, excisedfrom the gel, and purified using a QIAQUICK® Gel Extraction Kit (QIAGENInc., Valencia, Calif., USA). The gene fragment and the digested vectorwere combined together in a reaction resulting in the expression plasmidpAG66, in which transcription of the Family GH61N gene was under thecontrol of the NA2-tpi promoter. The recombination reaction (20 μl) wascomposed of 1× IN-FUSION™ Buffer, 1× BSA, 1μl of IN-FUSION™ enzyme(diluted 1:10), 186 ng of pAlLo2 digested with Nco I and Pac I, and 96.6ng of the Thielavia terrestris GH61N purified PCR product. The reactionwas incubated at 37° C. for 15 minutes followed by 15 minutes at 50° C.The reaction was diluted with 40 μl of TE buffer and 2.5 μl of thediluted reaction was used to transform E. coli Top10 Competent cells(Stratagene, La Jolla, Calif., USA). An E. coli transformant containingpAG66 (GH61N gene) was identified by restriction enzyme digestion andplasmid DNA was prepared using a BIOROBOT® 9600.

The same 1.1 kb Thielavia terrestris gh61n PCR fragment was also clonedinto pCR®2.1-TOPO vector using a TOPO® TA CLONING® Kit, to generatepAG68. The Thielavia terrestris gh61n insert was confirmed by DNAsequencing. E. coli pAG68 was deposited with the Agricultural ResearchService Patent Culture Collection, Northern Regional Research Center,Peoria, Ill., USA, on Sep. 18, 2009 and assigned accession number NRRLB-50320.

Example 18 Characterization of the Thielavia terrestris Genomic SequenceEncoding a Family GH61N Polypeptide having Cellulolytic EnhancingActivity

The nucleotide sequence (SEQ ID NO: 9) and deduced amino acid sequence(SEQ ID NO: 10) of the Thielavia terrestris GH61N polypeptide havingcellulolytic enhancing activity are shown in FIG. 8. The genomicpolynucleotide is 1107 bp, including the stop codon, and encodes apolypeptide of 368 amino acids. The % G+C content of the full-lengthcoding sequence and the mature coding sequence is 68.1% and 68.3%,respectively. Using the SignalP software program (Nielsen et al., 1997,supra), a signal peptide of 21 residues was predicted. The predictedmature protein contains 347 amino acids with a molecular mass of 35.0kDa.

Analysis of the deduced amino acid sequence of the GH61N polypeptidehaving cellulolytic enhancing activity with the Interproscan program(Mulder et al., 2007, Nucleic Acids Res. 35: D224-D228) showed that theGH61N polypeptide contained the sequence signature of glycosidehydrolase Family 61 (InterPro accession IPR005103). This sequencesignature was found from approximately residues 1 to 221 of the maturepolypeptide (Pfam accession PF03443).

A comparative pair wise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, supra) as implemented in the Needle program of EMBOSS with gapopen penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62matrix. The alignment showed that the deduced amino acid sequence of theThielavia terrestris GH61N mature polypeptide shares 72.3% identity(excluding gaps) to the deduced amino acid sequence of another predictedFamily 61 glycoside hydrolase protein from Aspergillus niger (UniProtaccession number A2QZE1).

Example 19 Expression of the Thielavia terrestris Genomic DNA EncodingFamily GH61N Polypeptides having Cellulolytic Enhancing Activity inAspergillus oryzae JaL355

Aspergillus oryzae JaL355 protoplasts were prepared according to themethod of Christensen et al., 1988, supra, which were transformed with 5pg of pAG43. Three transformants were isolated to individual PDA plates.

Plugs were taken from the original transformation plate of each of thethree transformants and added separately to 1 ml of M410 medium in 24well plates, which were incubated at 34° C. Five days after incubation,7.5 μl of supernatant from each culture was analyzed using CRITERION®stain-free, 8-16% gradient SDS-PAGE, (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA) according to the manufacturer's instructions.SDS-PAGE profiles of the cultures showed that several transformants hadnew major bands of approximately 70 kDa and 35 kDa.

Confluent PDA plates of two of the transformants were washed with 5 mlof 0.01% TWEEN® 20 and inoculated into five 500 ml Erlenmeyer flaskcontaining 100 ml of M410 medium and incubated to generate broth forcharacterization of the enzyme. The flasks were harvested on days 3 and5 and filtered using a 0.22 μm stericup suction filter (Millipore,Bedford, Mass., USA).

Example 20 Hydrolysis of Pretreated Corn Stover is Enhanced by Thielaviaterrestris GH61N Polypeptide having Cellulolytic Enhancing Activity

Culture broth was prepared as described in Example 19 and concentratedapproximately 20-fold using an Amicon ultrafiltration device (Millipore,Bedford, Mass, USA, 10 kDa polyethersulfone membrane, 40 psi, 4° C.).Protein concentration was estimated by densitometry following SDS-PAGEand Coomassie blue staining. Corn stover was pretreated and prepared asan assay substrate as described in WO 2005/074647 to generate pretreatedcorn stover (PCS). The base cellulase mixture used to assay enhancingactivity was prepared from Trichoderma reesei strain SMA135 (WO2008/057637).

Hydrolysis of PCS was conducted using 1.6 ml deep-well plates (Axygen,Santa Clara, Calif.) using a total reaction volume of 1.0 ml and a PCSconcentration of 50 mg/ml in 1 mM manganese sulfate-50 mM sodiumacetate, pH 5.0. The T. terrestris polypeptide (GH61N) was separatelyadded to the base cellulase mixture at concentrations ranging from 0 to100% of the protein concentration of the base cellulase mixture.Incubation was at 50° C. for 72 hours. Assays were performed intriplicate. Aliquots were centrifuged, and the supernatant liquid wasfiltered by centrifugation (MULTISCREEN® HV 0.45 μm, Millipore,Billerica, Mass., USA) at 3000 rpm for 10 minutes using a platecentrifuge (SORVALL® RT7, Thermo Fisher Scientific, Waltham, Mass.,USA). When not used immediately, filtered hydrolysate aliquots werefrozen at −20° C. Sugar concentrations of samples diluted in 0.005 MH₂SO₄ with 0.05% w/w benzoic acid were measured after elution by 0.005 MH₂SO₄ with 0.05% w/w benzoic acid at a flow rate of 0.6 ml/minute from a4.6×250 mm AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules,Calif., USA) at 65° C. with quantitation by integration of glucose andcellobiose signals from refractive index detection (CHEMSTATION®,AGILENT® 1100 HPLC, Agilent Technologies, Santa Clara, Calif., USA)calibrated by pure sugar samples (Absolute Standards Inc., Hamden,Conn., USA). The resultant equivalents were used to calculate thepercentage of cellulose conversion for each reaction. The degree ofcellulose conversion to glucose plus cellobiose sugars (conversion, %)was calculated using the following equation:

Conversion (%)=(glucose+cellobiose×1.053) (mg/ml)×100×162/(Cellulose(mg/ml)×180)=(glucose+cellobiose×1.053) (mg/ml)×100/(Cellulose(mg/ml)×1.111)

In this equation the factor 1.111 reflects the weight gain in convertingcellulose to glucose, and the factor 1.053 reflects the weight gain inconverting cellobiose to glucose. Cellulose in PCS was determined by alimit digest of PCS to release glucose and cellobiose.

The results of adding increasing amounts of Thielavia terrestrispolypeptide separately to the base cellulase mix are shown in FIG. 9.Addition of the T. terrestris GH61N provided a maximum stimulatorybenefit of 1.30 at an addition percentage of 50%.

Example 21 Construction of an Aspergillus oryzae Expression Vector forthe Thielavia terrestris Family GH610 Gene

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Thielavia terrestris Family GH61O gene from the genomic DNAprepared in Example 2. An IN-FUSION™ Cloning Kit was used to clone thefragment directly into the expression vector, pAlLo2, without the needfor restriction digests and ligation.

Forward primer: (SEQ ID NO: 33) 5′-ACTGGATTTACCATGCCGCCCGCACTCCCTCA-3′Reverse primer: (SEQ ID NO: 34)5′-TCACCTCTAGTTAATTAACTAACCCCGCCGATCATACC-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAlLo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 1 μg of Thielavia terrestris genomic DNA, 1× ADVANTAGE®GC-Melt LA Buffer, 1μl of 10 mM blend of dATP, dTTP, dGTP, and dCTP, and1.25 units of ADVANTAGE® GC Genomic LA Polymerase Mix, in a final volumeof 25 μl. The amplification conditions were one cycle at 94° C. for 1minute; and 30 cycles each at 94° C. for 30 seconds, 60.5° C. for 30seconds, and 72° C. for 1 minute. The heat block was then held at 72° C.for 5 minutes followed by a 4° C. soak cycle.

The reaction products were isolated on a 1.0% agarose gel using TAEbuffer where as approximately 1 kb product band was excised from the geland purified using a MINELUTE® Gel Extraction Kit according to themanufacturer's instructions.

The fragment was then cloned into pAlLo2 using an IN-FUSION™ CloningKit. The vector was digested with Nco I and Pac I. The fragment waspurified by gel electrophoresis and a QIAQUICK® Gel Extraction Kit. Thegene fragment and the digested vector were combined together in areaction resulting in the expression plasmid pAG67, in whichtranscription of the Family GH61O gene was under the control of theNA2-tpi promoter. The recombination reaction (20 μl) was composed of 1×IN-FUSION™ Buffer, 1× BSA, 1μl of IN-FUSION™ enzyme (diluted 1:10), 186ng of pAlLo2 digested with Nco I and Pac I, and 90.6 ng of the Thielaviaterrestris GH610 purified PCR product. The reaction was incubated at 37°C. for 15 minutes followed by 15 minutes at 50° C. The reaction wasdiluted with 40 μl of TE buffer and 2.5 μl of the diluted reaction wasused to transform E. coli Top10 Competent cells. An E. coli transformantcontaining pAG67 (GH61O gene) was identified by restriction enzymedigestion and plasmid DNA was prepared using a BIOROBOT® 9600.

The same 1 bp Thielavia terrestris gh61o PCR fragment was also clonedinto pCR®2.1-TOPO vector using a TOPO® TA CLONING® Kit, to generatepAG69. The Thielavia terrestris gh61o insert was confirmed by DNAsequencing. E. coli pAG69 was deposited with the Agricultural ResearchService Patent Culture Collection, Northern Regional Research Center,Peoria, Ill., USA, on Sep. 18, 2009 and assigned accession number NRRLB-50321.

Example 22 Characterization of the Thielavia terrestris Genomic SequenceEncoding a Family GH61O Polypeptide having Cellulolytic EnhancingActivity

The nucleotide sequence (SEQ ID NO: 11) and deduced amino acid sequence(SEQ ID NO: 12) of the Thielavia terrestris GH61O polypeptide havingcellulolytic enhancing activity are shown in FIG. 10. The genomicpolynucleotide is 993 bp, including the stop codon, and encodes apolypeptide of 330 amino acids. The % G+C content of the full-lengthcoding sequence and the mature coding sequence is 69.4% for both. Usingthe SignalP software program (Nielsen et al., 1997, supra), a signalpeptide of 24 residues was predicted. The predicted mature proteincontains 306 amino acids with a molecular mass of 32.1 kDa.

Analysis of the deduced amino acid sequence of the GH61O polypeptidehaving cellulolytic enhancing activity with the Interproscan program(Mulder et al., 2007, supra) showed that the GH61O polypeptide containedthe sequence signature of glycoside hydrolase Family 61 (InterProaccession IPR005103). This sequence signature was found fromapproximately residues 1 to 245 of the mature polypeptide (Pfamaccession PF03443).

A comparative pair wise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, supra) as implemented in the Needle program of EMBOSS with gapopen penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62matrix. The alignment showed that the deduced amino acid sequence of theThielavia terrestris GH61O mature polypeptide shares 56.5% identity(excluding gaps) to the deduced amino acid sequence of another predictedFamily 61 glycoside hydrolase protein from Podospora anserina (UniProtaccession number B2AVC8).

Example 23 Construction of an Aspergillus oryzae Expression Vector forthe Thielavia terrestris Family GH61P Gene

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Thielavia terrestris Family GH61P gene from the genomic DNAprepared in Example 2. An IN-FUSION™ Cloning Kit was used to clone thefragment directly into the expression vector, pAlLo2 (WO 2005/074647),without the need for restriction digests and ligation.

Forward primer: (SEQ ID NO: 35)5′-ACTGGATTTACCATGAAGACATTCACCGCCCTCCTG-3′ Reverse primer:(SEQ ID NO: 36) 5′-TCACCTCTAGTTAATTAATCAGCAAGTAAAGACCGCCG-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAlLo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 1 μg of Thielavia terrestris genomic DNA, 1× ADVANTAGE®GC-Melt LA Buffer, 1μl of 10 mM blend of dATP, dTTP, dGTP, and dCTP, and1.25 units of ADVANTAGE® GC Genomic LA Polymerase Mix, in a final volumeof 25 μl. The amplification conditions were one cycle at 94° C. for 1minute; and 30 cycles each at 94° C. for 30 seconds, 58.5° C. for 30seconds, and 72° C. for 1.5 minutes. The heat block was then held at 72°C. for 5 minutes followed by a 4° C. soak cycle.

The reaction products were isolated on a 1.0% agarose gel using TAEbuffer where an approximately 1.2 kb product band was excised from thegel and purified using a MINELUTE® Gel Extraction Kit according to themanufacturer's instructions.

The fragment was then cloned into pAlLo2 using an IN-FUSION™ CloningKit. The vector was digested with Nco I and Pac I. The fragment waspurified by gel electrophoresis and QIAQUICK® Gel Extraction Kit. Thegene fragment and the digested vector were combined together in areaction resulting in the expression plasmid pAG70, in whichtranscription of the Family GH61P gene was under the control of theNA2-tpi promoter. The recombination reaction (10 μl) was composed of 1×IN-FUSION™ Buffer, 1× BSA, 0.5 μl of IN-FUSION™ enzyme (diluted 1:10),93 ng of pAlLo2 digested with Nco I and Pac I, and 2 μl of the Thielaviaterrestris GH61P purified PCR product. The reaction was incubated at 37°C. for 15 minutes followed by 15 minutes at 50° C. The reaction wasdiluted with 40 μl of TE buffer and 2.5 μl of the diluted reaction wasused to transform E. coli Top10 Competent cells. An E. coli transformantcontaining pAG70 (GH61P gene) was identified by restriction enzymedigestion and plasmid DNA was prepared using a BIOROBOT® 9600.

The same 1.2 kb Thielavia terrestris gh61p PCR fragment was also clonedinto pCR02.1-TOPO vector using a TOPO® TA CLONING® Kit, to generatepAG75. The Thielavia terrestris gh61p insert was confirmed by DNAsequencing. E. coli pAG75 was deposited with the Agricultural ResearchService Patent Culture Collection, Northern Regional Research Center,Peoria, Ill., USA, on Sep. 18, 2009 and assigned accession number NRRLB-50322.

Example 24 Characterization of the Thielavia terrestris Genomic SequenceEncoding a Family GH61P polypeptide having Cellulolytic EnhancingActivity

The nucleotide sequence (SEQ ID NO: 13) and deduced amino acid sequence(SEQ ID NO: 14) of the Thielavia terrestris GH61P polypeptide havingcellulolytic enhancing activity are shown in FIG. 11. The genomicpolynucleotide is 1221 bp, including the stop codon, and the codingsequence is interrupted by three introns of 231, 75, and 96 bp. Thepredicted coding sequence encodes a polypeptide of 236 amino acids. The% G+C content of the full-length coding sequence (including introns) andthe mature coding sequence is 60.2% and 59.8%, respectively. Using theSignalP software program (Nielsen et al., 1997, supra), a signal peptideof 16 residues was predicted. The predicted mature protein contains 220amino acids with a molecular mass of 23.6 kDa.

Analysis of the deduced amino acid sequence of the GH61P polypeptidehaving cellulolytic enhancing activity with the Interproscan program(Mulder et al., 2007, supra) showed that the GH61P polypeptide containedthe sequence signature of glycoside hydrolase Family 61 (InterProaccession IPR005103). This sequence signature was found fromapproximately residues 1 to 212 of the mature polypeptide (Pfamaccession PF03443).

A comparative pair wise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, supra) as implemented in the Needle program of EMBOSS with gapopen penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62matrix. The alignment showed that the deduced amino acid sequence of theThielavia terrestris GH61P mature polypeptide shares 80.3% identity(excluding gaps) to the deduced amino acid sequence of another predictedFamily 61 glycoside hydrolase protein from Neurospora crassa (UniProtaccession number Q7SA19).

Example 25 Construction of an Aspergillus oryzae Expression Vector forthe Thielavia terrestris Family GH61R Gene

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Thielavia terrestris Family GH61R gene from the genomic DNAprepared in Example 2. An IN-FUSION™ Cloning Kit was used to clone thefragment directly into the expression vector, pAlLo2 (WO 2005/074647),without the need for restriction digests and ligation.

Forward primer: (SEQ ID NO: 37)5′-ACTGGATTTACCATGGCCTTGCTGCTCTTGGCAGGC-3′ Reverse primer:(SEQ ID NO: 38) 5′-TCACCTCTAGTTAATTAATCACCCATCCCATATCGGCC-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAlLo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 1 μg of Thielavia terrestris genomic DNA, 1× ADVANTAGE®GC-Melt LA Buffer, 1μl of 10 mM blend of dATP, dTTP, dGTP, and dCTP, and1.25 units of ADVANTAGE® GC Genomic LA Polymerase Mix, in a final volumeof 25 μl. The amplification conditions were one cycle at 94° C. for 1minute; and 30 cycles each at 94° C. for 30 seconds, 59.4° C. for 30seconds, and 72° C. for 1.5 minutes. The heat block was then held at 72°C. for 5 minutes followed by a 4° C. soak cycle.

The reaction products were isolated on a 1.0% agarose gel using TAEbuffer where as approximately 1 kb product band was excised from the geland purified using a MINELUTE® Gel Extraction Kit according to themanufacturer's instructions.

The fragment was then cloned into pAlLo2 using an IN-FUSION™ CloningKit. The vector was digested with Nco I and Pac I. The fragment waspurified by gel electrophoresis and QIAQUICK® Gel Extraction Kit. Thegene fragment and the digested vector were combined together in areaction resulting in the expression plasmid pAG71, in whichtranscription of the Family GH61R gene was under the control of theNA2-tpi promoter. The recombination reaction (10 μl) was composed of 1×IN-FUSION™ Buffer, 1× BSA, 0.5 μl of IN-FUSION™ enzyme (diluted 1:10),93 ng of pAlLo2 digested with Nco I and Pac I, and 2 μl of the Thielaviaterrestris GH61R purified PCR product. The reaction was incubated at 37°C. for 15 minutes followed by 15 minutes at 50° C. The reaction wasdiluted with 40 μl of TE buffer and 2.5 μl of the diluted reaction wasused to transform E. coli Top10 Competent cells. An E. coli transformantcontaining pAG71 (GH61R gene) was identified by restriction enzymedigestion and plasmid DNA was prepared using a BIOROBOT® 9600.

The same 1 kb Thielavia terrestris gh61 r PCR fragment was also clonedinto pCR®2.1-TOPO vector using a TOPO® TA CLONING® Kit, to generatepAG76. The Thielavia terrestris gh6lrinsert was confirmed by DNAsequencing. E. coli pAG76 was deposited with the Agricultural ResearchService Patent Culture Collection, Northern Regional Research Center,Peoria, Ill., USA, on Sep. 18, 2009 and assigned accession number NRRLB-50323.

Example 26 Characterization of the Thielavia terrestris Genomic SequenceEncoding a Family GH61R Polypeptide having Cellulolytic EnhancingActivity

The nucleotide sequence (SEQ ID NO: 15) and deduced amino acid sequence(SEQ ID NO: 16) of the Thielavia terrestris GH61R polypeptide havingcellulolytic enhancing activity are shown in FIG. 12. The genomicpolynucleotide is 933 bp, including the stop codon, and the codingsequence is interrupted by three introns of 72, 53, and 55 bp. Thepredicted coding sequence encodes a polypeptide of 250 amino acids. The% G+C content of the full-length coding sequence (including introns) andthe mature coding sequence is 61.8% and 61.6%, respectively. Using theSignalP software program (Nielsen et al., 1997, supra), a signal peptideof 18 residues was predicted. The predicted mature protein contains 232amino acids with a molecular mass of 26.0 kDa.

Analysis of the deduced amino acid sequence of the GH61R polypeptidehaving cellulolytic enhancing activity with the Interproscan program(Mulder et al., 2007, supra8) showed that the GH61R polypeptidecontained the sequence signature of glycoside hydrolase Family 61(InterPro accession IPR005103). This sequence signature was found fromapproximately residues 1 to 224 of the mature polypeptide (Pfamaccession PF03443).

A comparative pair wise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, supra) as implemented in the Needle program of EMBOSS with gapopen penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62matrix. The alignment showed that the deduced amino acid sequence of theThielavia terrestris GH61R mature polypeptide shares 72.8% identity(excluding gaps) to the deduced amino acid sequence of another predictedFamily 61 glycoside hydrolase protein from Chrysosporium lucknowense(GeneSeqP accession number AWI36233).

Example 27 Construction of an Aspergillus oryzae Expression Vector forthe Thielavia terrestris Family GH61S Gene

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Thielavia terrestris Family GH61S gene from the genomic DNAprepared in Example 2. An IN-FUSION™ Cloning Kit was used to clone thefragment directly into the expression vector, pAlLo2, without the needfor restriction digests and ligation.

Forward primer: (SEQ ID NO: 39)5′-ACTGGATTTACCATGATGCCGTCCCTTGTTCGCTTC-3′ Reverse primer:(SEQ ID NO: 40) 5′-TCACCTCTAGTTAATTAATCAACCATGTCTCCTGTCCC-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAlLo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 1 μg of Thielavia terrestris genomic DNA, 1× ADVANTAGE®GC-Melt LA Buffer, 1μl of 10 mM blend of dATP, dTTP, dGTP, and dCTP, and1.25 units of ADVANTAGE® GC Genomic LA Polymerase Mix, in a final volumeof 25 μl. The amplification conditions were one cycle at 94° C. for 1minute; and 30 cycles each at 94° C. for 30 seconds, 58.5° C. for 30seconds, and 72° C. for 1.5 minutes. The heat block was then held at 72°C. for 5 minutes followed by a 4° C. soak cycle.

The reaction products were isolated on a 1.0% agarose gel using TAEbuffer where as approximately 1.3 kb product band was excised from thegel and purified using a MINELUTE® Gel Extraction Kit according to themanufacturer's instructions.

The fragment was then cloned into pAlLo2 using an IN-FUSION™ CloningKit. The vector was digested with Nco I and Pac I. The fragment waspurified by gel electrophoresis and QIAQUICK® Gel Extraction Kit. Thegene fragment and the digested vector were combined together in areaction resulting in the expression plasmid pAG72, in whichtranscription of the Family GH61S gene was under the control of theNA2-tpi promoter. The recombination reaction (10 μl) was composed of 1×IN-FUSION™ Buffer, 1× BSA, 0.5 μl of IN-FUSION™ enzyme (diluted 1:10),93 ng of pAlLo2 digested with Nco I and Pac I, and 2 μl of the Thielaviaterrestris GH61S purified PCR product. The reaction was incubated at 37°C. for 15 minutes followed by 15 minutes at 50° C. The reaction wasdiluted with 40 μl of TE buffer and 2.5 μl of the diluted reaction wasused to transform E. coli Top10 Competent cells. An E. coli transformantcontaining pAG72 (GH61S gene) was identified by restriction enzymedigestion and plasmid DNA was prepared using a BIOROBOT® 9600.

The same 1.3 kb Thielavia terrestris gh61s PCR fragment was also clonedinto pCR®2.1-TOPO vector using a TOPO® TA CLONING® Kit, to generatepAG77. The Thielavia terrestris gh61s insert was confirmed by DNAsequencing. E. coli pAG77 was deposited with the Agricultural ResearchService Patent Culture Collection, Northern Regional Research Center,Peoria, Ill., USA, on Sep. 18, 2009 and assigned accession number NRRLB-50324.

Example 28 Characterization of the Thielavia terrestris Genomic SequenceEncoding a Family GH61S Polypeptide having Cellulolytic EnhancingActivity

The nucleotide sequence (SEQ ID NO: 17) and deduced amino acid sequence(SEQ ID NO: 18) of the Thielavia terrestris GH61S polypeptide havingcellulolytic enhancing activity are shown in FIG. 13. The genomicpolynucleotide is 1584 bp, including the stop codon, and the codingsequence is interrupted by two introns of 64 and 83 bp. The predictedcoding sequence encodes a polypeptide of 478 amino acids. The % G+Ccontent of the full-length coding sequence (including introns) and themature coding sequence is 63.9% and 64.0%, respectively. Using theSignalP software program (Nielsen et al., 1997, supra), a signal peptidewas predicted but its exact location was ambiguous. The vast majority ofGH61 mature polypeptides begin with a histidine residue, and thereforethe most likely signal peptide is from residues 1 to 22. The predictedmature protein contains 456 amino acids with a molecular mass of 48.7kDa.

Analysis of the deduced amino acid sequence of the GH61S polypeptidehaving cellulolytic enhancing activity with the Interproscan program(Mulder et al., 2007, supra) showed that the GH61S polypeptide containedthe sequence signature of glycoside hydrolase Family 61 (InterProaccession IPR005103). This sequence signature was found fromapproximately residues 108 to 222 of the mature polypeptide (Pfamaccession PF03443).

A comparative pair wise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, supra) as implemented in the Needle program of EMBOSS with gapopen penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62matrix. The alignment showed that the deduced amino acid sequence of theThielavia terrestris GH61S mature polypeptide shares 65.1% identity(excluding gaps) to the deduced amino acid sequence of another predictedFamily 61 glycoside hydrolase protein from Chaetomium globosum (UniProtaccession number Q2GZM2).

Example 29 Construction of an Aspergillus oryzae Expression Vector forthe Thielavia terrestris Family GH61T Gene

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Thielavia terrestris Family GH61T gene from the genomic DNAprepared in Example 2. An IN-FUSION™ Cloning Kit was used to clone thefragment directly into the expression vector, pAlLo2, without the needfor restriction digests and ligation.

Forward primer: (SEQ ID NO: 41) 5′-ACTGGATTTACCATGCAGCTCCTCGTGGGCTT-3′Reverse primer: (SEQ ID NO: 42)5′-TCACCTCTAGTTAATTAATCAGCCACTCCACACCGGCG-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAlLo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 1 μg of Thielavia terrestris genomic DNA, 1× ADVANTAGE®GC-Melt LA Buffer, 1μl of 10 mM blend of dATP, dTTP, dGTP, and dCTP, and1.25 units of ADVANTAGE® GC Genomic LA Polymerase Mix, in a final volumeof 25 μl. The amplification conditions were one cycle at 94° C. for 1minute; and 30 cycles each at 94° C. for 30 seconds, 60.5° C. for 30seconds, and 72° C. for 1.5 minutes. The heat block was then held at 72°C. for 5 minutes followed by a 4° C. soak cycle.

The reaction products were isolated on a 1.0% agarose gel using TAEbuffer where as approximately 900 bp product band was excised from thegel and purified using a MINELUTE® Gel Extraction Kit according to themanufacturer's instructions.

The fragment was then cloned into pAlLo2 using an IN-FUSION™ CloningKit. The vector was digested with Nco I and Pac I. The fragment waspurified by gel electrophoresis and QIAQUICK® Gel Extraction Kit. Thegene fragment and the digested vector were combined together in areaction resulting in the expression plasmid pAG73, in whichtranscription of the Family GH61T gene was under the control of theNA2-tpi promoter. The recombination reaction (10 μl) was composed of 1×IN-FUSION™ Buffer, 1× BSA, 0.5 μl of IN-FUSION™ enzyme (diluted 1:10),93 ng of pAlLo2 digested with Nco I and Pac I, and 2 μl of the Thielaviaterrestris GH61T purified PCR product. The reaction was incubated at 37°C. for 15 minutes followed by 15 minutes at 50° C. The reaction wasdiluted with 40 μl of TE buffer and 2.5 μl of the diluted reaction wasused to transform E. coli Top10 Competent cells. An E. coli transformantcontaining pAG73 (GH61T gene) was identified by restriction enzymedigestion and plasmid DNA was prepared using a BIOROBOT® 9600.

The same 900 bp Thielavia terrestris gh61t PCR fragment was also clonedinto pCR02.1-TOPO vector using a TOPO® TA CLONING® Kit, to generatepAG78. The Thielavia terrestris gh6lt insert was confirmed by DNAsequencing. E. coli pAG78 was deposited with the Agricultural ResearchService Patent Culture Collection, Northern Regional Research Center,Peoria, Ill., USA, on Sep. 18, 2009 and assigned accession number NRRLB-50325.

Example 30 Characterization of the Thielavia terrestris Genomic SequenceEncoding a Family GH61T Polypeptide having Cellulolytic EnhancingActivity

The nucleotide sequence (SEQ ID NO: 19) and deduced amino acid sequence(SEQ ID NO: 20) of the Thielavia terrestris GH61T polypeptide havingcellulolytic enhancing activity are shown in FIG. 14. The genomicpolynucleotide is 868 bp, including the stop codon, and the codingsequence is interrupted by two introns of 76 and 99 bp. The predictedcoding sequence encodes a polypeptide of 230 amino acids. The % G+Ccontent of the full-length coding sequence (including introns) and themature coding sequence is 61.5% and 61.4%, respectively. Using theSignalP software program (Nielsen et al., 1997, supra), a signal peptideof 16 residues was predicted. The predicted mature protein contains 214amino acids with a molecular mass of 23.1 kDa.

Analysis of the deduced amino acid sequence of the GH61T polypeptidehaving cellulolytic enhancing activity with the Interproscan program(Mulder et al., 2007, supra) showed that the GH61T polypeptide containedthe sequence signature of glycoside hydrolase Family 61 (InterProaccession IPR005103). This sequence signature was found fromapproximately residues 71 to 197 of the mature polypeptide (Pfamaccession PF03443).

A comparative pair wise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, supra) as implemented in the Needle program of EMBOSS with gapopen penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62matrix. The alignment showed that the deduced amino acid sequence of theThielavia terrestris GH61T mature polypeptide shares 87.4% identity(excluding gaps) to the deduced amino acid sequence of another predictedFamily 61 glycoside hydrolase protein from Chaetomium globosum (UniProtaccession number Q2GUTO).

Example 31 Construction of an Aspergillus oryzae Expression Vector forthe Thielavia terrestris Family GH61U Gene

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Thielavia terrestris Family GH61U gene from the genomic DNAprepared in Example 2. An IN-FUSION™ Cloning Kit was used to clone thefragment directly into the expression vector, pAlLo2, without the needfor restriction digests and ligation.

Forward primer: (SEQ ID NO: 43)5′-ACTGGATTTACCATGAAGCTGTACCTGGCGGCCTTT-3′ Reverse primer:(SEQ ID NO: 44) 5′-TCACCTCTAGTTAATTAATCAACCAGTCCACAGCGCTG-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAlLo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 1 μg of Thielavia terrestris genomic DNA, 1× ADVANTAGE®GC-Melt LA Buffer, 1μl of 10 mM blend of dATP, dTTP, dGTP, and dCTP, and1.25 units of ADVANTAGE® GC Genomic LA Polymerase Mix, in a final volumeof 25 μl. The amplification conditions were one cycle at 94° C. for 1minute; and 30 cycles each at 94° C. for 30 seconds, 58.5° C. for 30seconds, and 72° C. for 1.5 minutes. The heat block was then held at 72°C. for 5 minutes followed by a 4° C. soak cycle.

The reaction products were isolated on a 1.0% agarose gel using TAEbuffer where as approximately 1 kb product band was excised from the geland purified using a MINELUTE0 Gel Extraction Kit according to themanufacturer's instructions.

The fragment was then cloned into pAlLo2 using an IN-FUSION™ CloningKit. The vector was digested with Nco I and Pac I. The fragment waspurified by gel electrophoresis and QIAQUICK® Gel Extraction Kit. Thegene fragment and the digested vector were combined together in areaction resulting in the expression plasmid pAG74, in whichtranscription of the Family GH61U gene was under the control of theNA2-tpi promoter. The recombination reaction (10 μl) was composed of 1×IN-FUSION™ Buffer, 1× BSA, 0.5 μl of IN-FUSION™ enzyme (diluted 1:10),93 ng of pAlLo2 digested with Nco I and Pac I, and 2 μl of the Thielaviaterrestris GH61U purified PCR product. The reaction was incubated at 37°C. for 15 minutes followed by 15 minutes at 50° C. The reaction wasdiluted with 40 μl of TE buffer and 2.5 μl of the diluted reaction wasused to transform E. coli Top10 Competent cells. An E. coli transformantcontaining pAG74 (GH61U gene) was identified by restriction enzymedigestion and plasmid DNA was prepared using a BIOROBOT® 9600.

The same 1 kb Thielavia terrestris gh61 u PCR fragment was also clonedinto pCR02.1-TOPO vector using a TOPO® TA CLONING® Kit, to generatepAG79. The Thielavia terrestris gh61juinsert was confirmed by DNAsequencing. E. coli pAG79 was deposited with the Agricultural ResearchService Patent Culture Collection, Northern Regional Research Center,Peoria, Ill., USA, on September 18, 2009 and assigned accession numberNRRL B-50326.

Example 32 Characterization of the Thielavia terrestris Genomic SequenceEncoding a Family GH61U Polypeptide having Cellulolytic EnhancingActivity

The nucleotide sequence (SEQ ID NO: 21) and deduced amino acid sequence(SEQ ID NO: 22) of the Thielavia terrestris GH61U polypeptide havingcellulolytic enhancing activity are shown in FIG. 15. The genomicpolynucleotide is 1068 bp, including the stop codon, and the codingsequence is interrupted by four introns of 64, 52, 96 and 82 bp. Thepredicted coding sequence encodes a polypeptide of 257 amino acids. The% G+C content of the full-length coding sequence (including introns) andthe mature coding sequence is 59.7% and 59.3%, respectively. Using theSignalP software program (Nielsen et al., 1997, supra), a signal peptideof 19 residues was predicted. The predicted mature protein contains 238amino acids with a molecular mass of 26.6 kDa.

Analysis of the deduced amino acid sequence of the GH61U polypeptidehaving cellulolytic enhancing activity with the Interproscan program(Mulder et al., 2007, supra) failed to show that the GH61U polypeptidecontained the sequence signature of glycoside hydrolase Family 61(InterPro accession I PRO05103). However, a direct search against thePfam database produced a significant hit (e value of 4.3×10⁻⁸) to theGH61 family (Pfam accession PF03443). A comparative pair wise globalalignment of amino acid sequences was determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of EMBOSS with gap open penalty of 10,gap extension penalty of 0.5, and the EBLOSUM62 matrix. The alignmentshowed that the deduced amino acid sequence of the Thielavia terrestrisGH61U mature polypeptide shares 74.4% identity (excluding gaps) to thededuced amino acid sequence of another predicted Family 61 glycosidehydrolase protein from Chaetomium globosum (UniProt accession numberQ2HHT1).

Deposits of Biological Material

The following biological materials have been deposited under the termsof the Budapest Treaty with Agricultural Research Service Patent CultureCollection (NRRL), Northern Regional Research Center, 1815 UniversityStreet, Peoria, Ill., USA, and given the following accession numbers:

Deposit Accession Number Date of Deposit E. coli (pSMai216) NRRL B-50301Aug. 3, 2009 E. coli (pSMai217) NRRL B-50302 Aug. 3, 2009 E. coli(pSMai218) NRRL B-50303 Aug. 3, 2009 E. coli (pSMai213) NRRL B-50300Aug. 3, 2009 E. coli (pAG68) NRRL B-50320 Sep. 18, 2009 E. coli (pAG69)NRRL B-50321 Sep. 18, 2009 E. coli (pAG75) NRRL B-50322 Sep. 18, 2009 E.coli (pAG76) NRRL B-50323 Sep. 18, 2009 E. coli (pAG77) NRRL B-50324Sep. 18, 2009 E. coli (pAG78) NRRL B-50325 Sep. 18, 2009 E. coli (pAG79)NRRL B-50326 Sep. 18, 2009

The strains have been deposited under conditions that assure that accessto the cultures will be available during the pendency of this patentapplication to one determined by foreign patent laws to be entitledthereto. The deposits represent substantially pure cultures of thedeposited strains. The deposits are available as required by foreignpatent laws in countries wherein counterparts of the subjectapplication, or its progeny are filed. However, it should be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1-21. (canceled)
 22. A DNA construct or recombinant expression vectorcomprising an isolated polynucleotide comprising a nucleotide sequencethat encodes a GH61 polypeptide having cellulolytic enhancing activity,wherein the isolated polynucleotide is operably linked to one or moreheterologous control sequences that direct the production of thepolypeptide in an expression host, and wherein the GH61 polypeptidehaving cellulolytic enhancing activity is selected from: (a) a GH61polypeptide having at least 95% sequence identity to amino acids 20 to223 of SEQ ID NO: 8; (b) a GH61 polypeptide encoded by a polynucleotidethat hybridizes under very high stringency conditions with (i)nucleotides 58 to 974 of SEQ ID NO: 7, (ii) the cDNA sequence ofnucleotides 58 to 974 of SEQ ID NO: 7, or (iii) the full-lengthcomplement of (i) or (ii), wherein very high stringency conditions aredefined as prehybridization and hybridization at 42° C. in 5× SSPE, 0.3%SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50%formamide and washing three times each for 15 minutes using 2× SSC, 0.2%SDS at 70° C.; (c) a GH61 polypeptide encoded by a polynucleotide havingat least 99% sequence identity to nucleotides 58 to 974 of SEQ ID NO: 7,or a GH61 polypeptide encoded by the cDNA of nucleotides 58 to 974 ofSEQ ID NO: 7; and (d) an isolated fragment of a GH61 polypeptide of (a),(b), or (c) that has cellulolytic enhancing activity.
 23. The DNAconstruct or recombinant expression vector of claim 22, wherein the GH61polypeptide has at least 96% sequence identity to amino acids 20 to 223of SEQ ID NO:
 8. 24. The DNA construct or recombinant expression vectorof claim 22, wherein the GH61 polypeptide has at least 97% sequenceidentity to amino acids 20 to 223 of SEQ ID NO:
 8. 25. The DNA constructor recombinant expression vector of claim 22, wherein the GH61polypeptide has at least 98% sequence identity to amino acids 20 to 223of SEQ ID NO:
 8. 26. The DNA construct or recombinant expression vectorof claim 22, wherein the GH61 polypeptide has at least 99% sequenceidentity to amino acids 20 to 223 of SEQ ID NO:
 8. 27. The DNA constructor recombinant expression vector of claim 22, wherein the GH61polypeptide comprises amino acids 20 to 223 of SEQ ID NO: 8, or afragment thereof having cellulolytic enhancing activity.
 28. The DNAconstruct or recombinant expression vector of claim 22, wherein the GH61polypeptide consists of amino acids 20 to 223 of SEQ ID NO: 8, or afragment thereof having cellulolytic enhancing activity.
 29. The DNAconstruct or recombinant expression vector of claim 22, wherein the GH61polypeptide is encoded by a polynucleotide comprising nucleotides 58 to974 of SEQ ID NO: 7, or the cDNA of nucleotides 58 to 974 of SEQ ID NO:7.
 30. The DNA construct or recombinant expression vector of claim 22,wherein the GH61 polypeptide is encoded by a polynucleotide consistingof nucleotides 58 to 974 of SEQ ID NO: 7, or the cDNA of nucleotides 58to 974 of SEQ ID NO:
 7. 31. The DNA construct or recombinant expressionvector of claim 22, wherein the GH61 polypeptide is encoded by thepolynucleotide contained in pSMai213 which is contained in E. coli NRRLB-50300 deposited with the Northern Regional Research Center of theAgriculture Research Service Patent Culture Collection.
 32. Arecombinant host cell comprising the DNA construct or recombinantexpression vector of claim
 22. 33. A method of producing GH61polypeptide having cellulolytic enhancing activity, said methodcomprising: (a) cultivating the recombinant host cell of claim 32 underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.
 34. A DNA construct comprising a nucleicacid encoding a signal peptide comprising amino acids acids 1 to 19 ofSEQ ID NO: 8, wherein the nucleic acid is operably linked to aheterologous nucleic acid encoding a protein.
 35. A recombinant hostcell comprising a nucleic acid encoding a protein, wherein the nucleicacid is operably linked to an isolated polynucleotide encoding a signalpeptide comprising amino acids 1 to 19 of SEQ ID NO: 8, wherein thenucleic acid is foreign to the polynucleotide encoding the signalpeptide.
 36. A method of producing a protein, said method comprising:(a) cultivating the recombinant host cell of claim 35 under conditionsconducive for production of the protein; and (b) recovering the protein.37. A transgenic plant, plant part or plant cell transformed with apolynucleotide encoding a GH61 polypeptide having cellulolytic enhancingactivity, wherein the polypeptide comprises a signal peptide directingthe polypeptide into the secretory pathway, and wherein the GH61polypeptide having cellulolytic enhancing activity is selected from: (a)a GH61 polypeptide having at least 95% sequence identity to amino acids20 to 223 of SEQ ID NO: 8; (b) a GH61 polypeptide encoded by apolynucleotide that hybridizes under very high stringency conditionswith (i) nucleotides 58 to 974 of SEQ ID NO: 7, (ii) the cDNA sequenceof nucleotides 58 to 974 of SEQ ID NO: 7, or (iii) the full-lengthcomplement of (i) or (ii), wherein very high stringency conditions aredefined as prehybridization and hybridization at 42° C. in 5× SSPE, 0.3%SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50%formamide and washing three times each for 15 minutes using 2× SSC, 0.2%SDS at 70° C.; (c) a GH61 polypeptide encoded by a polynucleotide havingat least 99% sequence identity to nucleotides 58 to 974 of SEQ ID NO: 7,or a GH61 polypeptide encoded by the cDNA of nucleotides 58 to 974 ofSEQ ID NO: 7; and (d) an isolated fragment of a GH61 polypeptide of (a),(b), or (c) that has cellulolytic enhancing activity.
 38. A method ofproducing a GH61 polypeptide having cellulolytic enhancing activity,said method comprising: (a) cultivating the transgenic plant or plantcell of claim 37 under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.
 39. A method fordegrading a cellulosic material comprising: (i) treating the cellulosicmaterial with an enzyme composition comprising a GH61 polypeptide havingcellulolytic enhancing activity, and (ii) recovering the degradedcellulosic material; wherein the GH61 polypeptide having cellulolyticenhancing activity is selected from: (a) a GH61 polypeptide having atleast 95% sequence identity to amino acids 20 to 223 of SEQ ID NO: 8;(b) a GH61 polypeptide encoded by a polynucleotide that hybridizes undervery high stringency conditions with (i) nucleotides 58 to 974 of SEQ IDNO: 7, (ii) the cDNA sequence of nucleotides 58 to 974 of SEQ ID NO: 7,or (iii) the full-length complement of (i) or (ii), wherein very highstringency conditions are defined as prehybridization and hybridizationat 42° C. in 5× SSPE, 0.3% SDS, 200 micrograms/ml sheared and denaturedsalmon sperm DNA, and 50% formamide and washing three times each for 15minutes using 2× SSC, 0.2% SDS at 70° C.; (c) a GH61 polypeptide encodedby a polynucleotide having at least 99% sequence identity to nucleotides58 to 974 of SEQ ID NO: 7, or a GH61 polypeptide encoded by the cDNA ofnucleotides 58 to 974 of SEQ ID NO: 7; and (d) an isolated fragment of aGH61 polypeptide of (a), (b), or (c) that has cellulolytic enhancingactivity.
 40. A method for producing a fermentation product, said methodcomprising: (i) saccharifying a cellulosic material with an enzymecomposition comprising a GH61 polypeptide having cellulolytic enhancingactivity; (ii) fermenting the saccharified cellulosic material with oneor more fermenting microorganisms to produce the fermentation product;and (ii) recovering the fermentation product from the fermentation;wherein the GH61 polypeptide having cellulolytic enhancing activity isselected from: (a) a GH61 polypeptide having at least 95% sequenceidentity to amino acids 20 to 223 of SEQ ID NO: 8; (b) a GH61polypeptide encoded by a polynucleotide that hybridizes under very highstringency conditions with (i) nucleotides 58 to 974 of SEQ ID NO: 7,(ii) the cDNA sequence of nucleotides 58 to 974 of SEQ ID NO: 7, or(iii) the full-length complement of (i) or (ii), wherein very highstringency conditions are defined as prehybridization and hybridizationat 42° C. in 5× SSPE, 0.3% SDS, 200 micrograms/ml sheared and denaturedsalmon sperm DNA, and 50% formamide and washing three times each for 15minutes using 2× SSC, 0.2% SDS at 70° C.; (c) a GH61 polypeptide encodedby a polynucleotide having at least 99% sequence identity to nucleotides58 to 974 of SEQ ID NO: 7, or a GH61 polypeptide encoded by the cDNA ofnucleotides 58 to 974 of SEQ ID NO: 7; and (d) an isolated fragment of aGH61 polypeptide of (a), (b), or (c) that has cellulolytic enhancingactivity.
 41. The method of claim 40, wherein steps (i) and (ii) areperformed simultaneously.
 42. A method of fermenting a cellulosicmaterial comprising: (i) fermenting the cellulosic material with one ormore fermenting microorganisms, wherein the cellulosic material issaccharified with an enzyme composition comprising a polypeptide havingcellulolytic enhancing activity, wherein the fermenting of thecellulosic material produces a fermentation product, and (ii) recoveringthe fermentation product from the fermentation; wherein the GH61polypeptide having cellulolytic enhancing activity is selected from: (a)a GH61 polypeptide having at least 95% sequence identity to amino acids20 to 223 of SEQ ID NO: 8; (b) a GH61 polypeptide encoded by apolynucleotide that hybridizes under very high stringency conditionswith (i) nucleotides 58 to 974 of SEQ ID NO: 7, (ii) the cDNA sequenceof nucleotides 58 to 974 of SEQ ID NO: 7, or (iii) the full-lengthcomplement of (i) or (ii), wherein very high stringency conditions aredefined as prehybridization and hybridization at 42° C. in 5× SSPE, 0.3%SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50%formamide and washing three times each for 15 minutes using 2× SSC, 0.2%SDS at 70° C.; (c) a GH61 polypeptide encoded by a polynucleotide havingat least 99% sequence identity to nucleotides 58 to 974 of SEQ ID NO: 7,or a GH61 polypeptide encoded by the cDNA of nucleotides 58 to 974 ofSEQ ID NO: 7; and (d) an isolated fragment of a GH61 polypeptide of (a),(b), or (c) that has cellulolytic enhancing activity.